Nucleic acids encoding isav polypeptides

ABSTRACT

Infectious Salmon Anemia Virus (ISAV) nucleic acid molecules and polypeptides are disclosed, as well as host cells and transgenic fish transformed by expression vectors containing such nucleic acids. The nucleic acid molecules can encode antigenic epitopes capable of eliciting an immune response in a host cell or animal, such as an immune response against ISAV, and the polypeptides themselves can be antigenic epitopes and also induce such an immune response.

FIELD

[0001] This invention relates to Infectious Salmon Anemia Virus (ISAV),more specifically to ISAV nucleic acid sequences and the peptides thesenucleic acids encode. This invention also relates to the use of ISAVpeptides in producing an immune response in fish.

BACKGROUND

[0002] Global aquaculture production is estimated at 39.4 million tonsannually, is worth $52.5 billion (US), and contributes over 20% of thetotal fish harvest. Although the United States contributes only 2% ofglobal production, the aquaculture industry in this country is gainingmomentum and importance. For example, farm-raised salmon are a prominentindustry in the Pacific Northwest and Maine.

[0003] As the natural fisheries provided by the open seas declineglobally, and the world's population is projected to grow to 8 billionpeople by 2025, cultured finfish products will be in increasing demandas an important protein source. Some of the factors that must besuccessfully accommodated to sustain the economic viability and increasethe productivity of finfish culture include maintaining adequate culturefacilities, complying with regulatory and environmental requirements andcountering the many infectious pathogens and diseases that can threatenfarmed populations of aquatic animals. Of these variables, the economicimpact of disease on cultured finfish operations has become increasinglyimportant. One of the primary means for raising finfish cultureefficiency is through the development of reliable treatments againstinfectious pathogens and thus improve the overall health of farmedspecies.

[0004] Infectious salmon anemia (ISA), formerly called HemorrhagicKidney Syndrome (HKS), has caused massive economic losses in theAtlantic salmon farming industry in Norway, Atlantic Canada, andScotland. Mortality from ISA disease is variable, ranging from 10% tomore than 50%. Clinical signs of the disease are apparent in Atlanticsalmon, but other salmonids can act as non-symptomatic reservoirs forthe virus. The pathological changes associated with ISA arecharacterized by severe anemia, leukopenia, ascites and hemorrhaging ofinternal organs with subsequent necrosis of hepatocytes and renalinterstitial cells. The infectious agent is an enveloped virus (ISAV)which replicates in endothelial cells in vivo and buds from the cellsurface. The virus has a single-stranded RNA genome consisting of 8segments with negative polarity, and the structural, morphological, andphysiochemical properties of the virus suggest that ISAV is related tomembers of the Orthomyxoviridae family (see, e.g., Falk, et al., J.Virol. 71:9016-23 (1997)).

[0005] ISA originally appeared in Norway in 1984 (Thorud and Djubvik,1988). In 1996 and 1998, the disease was diagnosed on fish farms inAtlantic Canada and Scotland, respectively. Subsequent to the appearanceof clinical disease in Canada, ISAV surveillance programs wereinstituted in New Brunswick. A central aspect of the Canadian ISAVmanagement approach involves the depopulation of ISAV-infected cagesthat are found through participation in the surveillance protocols. TheCanadian government and Canadian salmon producers themselves havedeveloped several compensation programs to offset losses fromeradication measures, which has helped lower the incidence of new casesof both virus and disease at previously negative marine sites. RecentCanadian outbreaks are currently confined to the Bay of Fundy area ofMaritime Canada. However, the Norwegian disease pattern has shown thatthe virus spreads from population to population principally by exposureto body fluids from infected fish, through untreated water coming fromfish processing plants or through shared equipment that hasn't beenproperly disinfected at marine sites. Thus, Atlantic salmon netpens atneighboring Maine marine sites are at considerable risk of encounteringISA virus.

[0006] Historically, the elimination of ISA disease in other countriesthrough the attempted eradication of ISA virus has proven to be futile.Given the many unknown factors involved in disease transmission,including ties between the ISA pathogen and wild reservoirs of virus,outright elimination of ISA and the virus (ISAV) does not appear to bean achievable goal. However, as shown over time in several otherinternational epizootics of ISA, mortality from ISA can be decreasedthrough the development of biosecurity protocols and good managementtechniques. Nonetheless, the development of effective treatments againstISAV remains a high priority for salmon producers in the U.S. andelsewhere.

[0007] Fish that survive ISA demonstrative a protective immune responseindicating that prophylactic treatment against ISA is possible. Wholekilled viral formulations have been shown to be effective against otherviral diseases of fish, but the disadvantage of such an approach is thatvirulent virus may remain in the formulation if extreme care is nottaken during the manufacturing process. Additionally, the immuneresponse conferred is often brief and may need to be boosted. Finally,killed virus formulations are prepared by growing virus in large amountsin cell culture or in the actual animal species, and either method isexpensive. Furthermore, if the titer of the amplified virus is low, thenachieving the appropriate antigenic dose within the final formulationrequires the addition of more virus and raises the cost of production.Thus, a need remains for an effective ISAV vaccine.

SUMMARY

[0008] ISAV nucleic acid molecules are disclosed. In some embodiments,the nucleic acid molecule has a sequence at least 70% identical to SEQID NO: 1, a nucleic acid sequence at least 85% identical to SEQ ID NO:3, or a nucleic acid sequence at least 85% identical to SEQ ID NO: 11,or a sequence consisting essentially of SEQ ID NO: 1, SEQ ID NO: 3, orSEQ ID NO: 11. In particular embodiments, the nucleic acid molecule isoperably linked to a heterologous nucleic acid, such as an expressioncontrol sequence. In one specific non-limiting example, the nucleic acidsequence is included in a vector.

[0009] Host cells and transgenic fish transformed by such nucleic acidsalso are disclosed. In some embodiments, the nucleic acid moleculeencodes an antigenic epitope capable of eliciting an immune response inthe cell or fish, such as an immune response against ISAV. Particularfish and fish cells include (but are not limited to) rainbow trout, cohosalmon, chinook salmon, amago salmon, chum salmon, sockeye salmon,Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout,catfish, tilapia, sea bream, seabass, flounder, or sturgeon.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph showing the results of an efficacy trial ofAtlantic salmon treated with whole killed ISAV and challenged with liveISAV.

[0011]FIG. 2 is a digital image of the results of SDS-PAGE analysis ofpurified ISAV proteins.

[0012]FIG. 3 is a graph illustrating the results of a humoral immuneresponse to whole killed ISAV in Atlantic salmon.

[0013]FIG. 4 is the amino acid sequence alignment of the RNA bindingdomain of NP from influenza virus A and B with the putative NP RNAbinding domain from ISA virus. This alignment was predicted using theClustal W system.

[0014]FIG. 5 is a graph illustrating the titration of ISAV-specificantibodies from Atlantic salmon infected with ISAV.

[0015]FIG. 6 is a graph illustrating the ISAV-specific antibodies insera obtained from Atlantic salmon infected with ISAV or rainbow troutinjected with a nucleic acid encoding an ISAV-specific protein.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0016] The nucleic acid sequences listed herein are shown using standardletter abbreviations for nucleotide bases. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

[0017] SEQ ID NO: 1 shows a 2.4 kbp nucleic acid fragment of ISAV(segment 1) with a partial open reading frame (orf) encoding the P1protein.

[0018] SEQ ID NO: 2 shows the partial amino acid sequence of the P1protein encoded by SEQ ID NO: 1.

[0019] SEQ ID NO: 3 shows a 2.4 kbp nucleic acid fragment of ISAV(segment 2) with a 2127 bp orf encoding the PB1 protein.

[0020] SEQ ID NO: 4 shows the amino acid sequence of the PB1 protein,measuring 709 aa, encoded by SEQ ID NO: 3.

[0021] SEQ ID NO: 5 shows a 2.2 kbp nucleic acid fragment of ISAV(segment 3) with a 1851 bp orf encoding the NP protein.

[0022] SEQ ID NO: 6 shows the amino acid sequence of the NP protein,measuring 617 aa, encoded by SEQ ID NO: 5.

[0023] SEQ ID NO: 7 shows a 1.9 kbp nucleic acid fragment of ISAV(segment 4) with a 1737 bp orf encoding the P2 protein.

[0024] SEQ ID NO: 8 shows the amino acid sequence of the P2 protein,measuring 579 aa, encoded by SEQ ID NO: 8.

[0025] SEQ ID NO: 9 shows a 1.6 kbp nucleic acid fragment of ISAV(segment 5) with a 1335 bp orf encoding the P3 protein.

[0026] SEQ ID NO: 10 shows the amino acid sequence of the P3 protein,measuring 445 aa, encoded by SEQ ID NO: 9.

[0027] SEQ ID NO: 11 shows a 1.5 kbp nucleic acid fragment of ISAV(segment 6) with an 1185 bp orf encoding the HA protein.

[0028] SEQ ID NO: 12 shows the amino acid sequence of the HA protein,measuring 395 aa, encoded by SEQ ID NO: 10.

[0029] SEQ ID NO: 13 shows a 1.3 kbp nucleic acid fragment of ISAV(segment 7) with a 771 bp orf encoding the P4 protein and a 441 bp orfencoding the P5 protein.

[0030] SEQ ID NO: 14 shows the amino acid sequence of the P4 protein,measuring 257 aa, encoded by SEQ ID NO: 13.

[0031] SEQ ID NO: 15 shows the amino acid sequence of the P5 protein,measuring 147 aa, also encoded by SEQ ID NO: 13.

[0032] SEQ ID NO: 16 shows a 1.0 kbp nucleic acid fragment of ISAV(segment 8) with a 705 bp orf encoding the P6 protein and a 552 bp orfencoding the P7 protein.

[0033] SEQ ID NO: 17 shows the amino acid sequence of the P6 protein,measuring 235 aa, encoded by SEQ ID NO: 16.

[0034] SEQ ID NO: 18 shows the amino acid sequence of the P7 protein,measuring 184 aa, also encoded by SEQ ID NO: 16.

DETIALED DESCRIPTION

[0035] Abbreviations

[0036] aa=ammo acid

[0037] bp=base pair

[0038] ISA=infectious salmon anemia

[0039] ISAV=infectious salmon anemia virus

[0040] kbp=kilo-base pair

[0041] orf=open reading frame

[0042] PCR=polymerase chain reaction

[0043] RT=reverse transcription

[0044] Terms

[0045] The following explanations of terms are provided in order tofacilitate review of the embodiments described herein. Explanations ofcommon terms also can be found in Rieger et al., Glossary of Genetics:Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991;Lewin, Nucleic acids VII, Oxford University Press: New York, 1999; andDictionary of Bioscience, Mcgraw-Hill: New York, 1997.

[0046] The singular forms “a,” “an,” and “the” refer to one or more thanone, unless the context clearly dictates otherwise. For example, theterm “comprising a nucleic acid” includes single or plural nucleic acidsand is considered equivalent to the phrase “comprising at least onenucleic acid.”

[0047] The term “or” refers to a single element of stated alternativeelements or a combination of two or more elements. For example, thephrase “a first nucleic acid or a second nucleic acid” refers to thefirst nucleic acid, the second nucleic acid, or both the first andsecond nucleic acids.

[0048] As used herein, “comprises” means “includes.” Thus, “comprising Aand B” means “including A and B,” without excluding additional elements.

[0049] The standard one- and three-letter nomenclature for amino acidresidues is used.

[0050] Amplification of a Nucleic Acid.

[0051] Any of several techniques that increases the number of copies ofa nucleic acid molecule. An example of amplification is the polymerasechain reaction (PCR), in which a sample containing the nucleic acid iscontacted with a pair of oligonucleotide primers under conditions thatallow for the hybridization of the primers to nucleic acid in thesample. The primers are extended under suitable conditions, dissociatedfrom the template, and then re-annealed, extended, and dissociated toamplify the number of copies of the nucleic acid. The amplificationproducts (called “amplicons”) can be further processed, manipulated, orcharacterized by (without limitation) electrophoresis, restrictionendonuclease digestion, hybridization, nucleic acid sequencing,ligation, or other techniques of molecular biology. Other examples ofamplification include strand displacement amplification, as disclosed inU.S. Pat. No. 5,744,311; transcription-free isothermal amplification, asdisclosed in U.S. Pat. No. 6,033,881; repair chain reactionamplification, as disclosed in WO 90/01069; ligase chain reactionamplification, as disclosed in European Patent Appl. 320 308; gapfilling ligase chain reaction amplification, as disclosed in U.S. Pat.No. 5,427,930; and NASBA™ RNA transcription-free amplification, asdisclosed in U.S. Pat. No. 6,025,134.

[0052] Conservative Amino-Acid Substitution.

[0053] Conservative amino acid substitutions in a polypeptide, such asan ISAV polypeptide, include those listed in Table 1 below. TABLE 1Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

[0054] Non-conservative substitutions are those that disrupt thesecondary, tertiary, or quaternary conformation of a polypeptide. Suchnon-conservative substitutions can result from changes in: (a) thestructure of the polypeptide backbone in the area of the substitution;(b) the charge or hydrophobicity of the polypeptide; or (c) the bulk ofan amino acid side chain. Substitutions generally expected to producethe greatest changes in polypeptide properties are those in which: (a) ahydrophilic residue is substituted for (or by) a hydrophobic residue;(b) a proline is substituted for (or by) any other residue; or (c) aresidue having a bulky side chain, for example, phenylalanine, issubstituted for (or by) one not having a side chain, for example,glycine. In particular embodiments, a residue having an electropositiveside chain, for example, lysyl, arginyl, or histadyl, is not substitutedfor (or by) an electronegative residue, for example, glutamyl oraspartyl.

[0055] Analog or Homolog.

[0056] An analog is a molecule that differs in chemical structure from aparent compound. A homolog differs by an increment in the chemicalstructure (such as a difference in the length of a nucleic acid or aminoacid chain), a molecular fragment, a structure that differs by one ormore functional groups, or a change in ionization.

[0057] Antigen.

[0058] A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes.

[0059] Animal.

[0060] A living, multi-cellular, vertebrate organism, including, forexample, mammals, birds, reptiles, and fish. The term “aquacultureanimal” includes all species suitable for aquaculture farming, such asfish, cephalopods, and crustaceans, including the specific speciesdescribed herein. Similarly, the term “subject” includes both human andveterinary subjects, such as aquaculture animals.

[0061] cDNA (Complementary DNA).

[0062] A piece of DNA lacking internal, non-coding segments (introns)and regulatory sequences that determine transcription. cDNA can besynthesized in a laboratory by reverse transcription from messenger RNAextracted from cells.

[0063] Complementarity.

[0064] A nucleic acid that performs a similar function to the sequenceto which it is complementary. The complementary sequence does not haveto confer replication competence in the same cell type to becomplementary, but merely confer replication competence in some celltype.

[0065] Delivery of Compositions.

[0066] For administration to animals, purified active compositions canbe administered alone or combined with an acceptable carrier.Preparations can contain one type of therapeutic molecule, or can becomposed of a combination of several types of therapeutic molecules. Thenature of the carrier will depend on the particular mode ofadministration being utilized. For instance, parenteral formulationsusually comprise injectable fluids that include physiologicallyacceptable fluids such as water, physiological saline, balanced saltsolutions, aqueous dextrose, glycerol or the like as a vehicle. Forsolid compositions (e.g., powder, pill, tablet, or capsule forms), whichcan be added to an aquaculture environment, conventional non-toxic solidcarriers can include, for example, mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,compositions to be administered to fish can contain minor amounts ofnon-toxic auxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

[0067] It is also contemplated that the nucleic acids could be deliveredto cells subsequently expressed by the host cell, for example throughthe use viral vectors, plasmid vectors, or liposomes administered tofish.

[0068] Compositions of the present invention can be administered by anymeans that achieve their intended purpose. Amounts and regimens for theadministration of the nucleic acids, or an active fragment thereof, canbe readily determined.

[0069] For use in treating viral infections, compositions areadministered in an amount effective to inhibit viral infection orprogression of an existing infection, or administered in an amounteffective to inhibit or alleviate a corresponding disease. In oneembodiment, infection is completely prevented.

[0070] Typical amounts initially administered would be those amountsadequate to achieve tissue concentrations at the site of action whichhave been found to achieve the desired effect in vitro. The compositionscan be administered to a host in vivo, for example through systemicadministration, such as intravenous, intramuscular, or intraperitonealadministration. The compositions also can be administeredintralesionally, through scarification of the skin, intrabuccaladministration, cutaneous particle bombardment, or by immersion in watercontaining a nucleic acid composition described herein (for uptake bythe fish). Additionally, the nucleic acid compositions can beadministered by encapsulation with a nanoparticle matrix composed of anucleic acid in methacrylic acid polymer, and an attenuated bacteria(such as Yersinia ruckeri, Edwardsiella ictaluri, Aeromonas salmonicida,or Vibrio anguillarum) carrying the nucleic acid for delivery byimmersion administration (see, e.g., U.S. Pat. No. 5,877,159, hereinincorporated by reference).

[0071] Effective doses for using compositions can vary depending on theseverity of the condition to be treated, the age and physiologicalcondition of the fish, mode of administration, and other relevantfactors. Thus, the final determination of the appropriate treatmentregimen can be made by someone at the site of the fish, such as anoperator or employee of an aquaculture facility. Typically, the doserange will be from about 1 μg/kg body weight to about 100 mg/kg bodyweight, such as about 10 μg/kg body weight to about 900 μg/kg bodyweight, or from about 50 μg/kg body weight to about 500 μg/kg bodyweight, or from about 50 μg/kg body weight to about 150 μg/kg bodyweight, such as about 100 μg/kg body weight. Nanogram quantities oftransforming DNA have been shown to be capable of inducing an immuneresponse in fish (see, e.g., Corbeil, S., et al., Vaccine 18(25):2817-24(2000), herein incorporated by reference).

[0072] The dosing schedule can vary from a single dosage to multipledosages given several times a day, once a day, once every few days, oncea week, once a month, annually, biannually, biennially, or any otherappropriate periodicity. The dosage schedule can depend on a number offactors, such as the species' or subject's sensitivity to thecomposition, the type and severity of infection, route ofadministration, and the volume of the container that contains the fish.In the case of a more aggressive disease, compositions can beadministered by alternate routes, including intramuscularly and byenvironmental uptake. Continuous administration also can be appropriatein some circumstances, for example, immersing fish or other aquacultureanimals in water containing the composition.

[0073] Hybridization Conditions.

[0074] “Stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 25% mismatch betweenthe hybridization probe and the target sequence. “Stringent conditions”can be broken down into particular levels of stringency for more precisemeasurement. Thus, as used herein, “moderate stringency” conditions arethose under which DNA molecules with more than 25% sequence variation(also termed “mismatch”) will not hybridize; conditions of “mediumstringency” are those under which DNA molecules with more than 15%mismatch will not hybridize, and conditions of “high stringency” arethose under which DNA sequences with more than 10% mismatch will nothybridize. Conditions of “very high stringency” are those under whichDNA sequences with more than 6% mismatch will not hybridize.

[0075] Hybridization.

[0076] Oligonucleotides hybridize by hydrogen bonding, which includesWatson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding betweencomplementary nucleotide units. For example, adenine and thymine arecomplementary nucleotides that pair through formation of hydrogen bonds.“Complementary” refers to sequence complementarity between twonucleotide units. For example, if a nucleotide unit at a certainposition of an oligonucleotide is capable of hydrogen bonding with anucleotide unit at the same position of a nucleic acid molecule, thenthe oligonucleotides are complementary to each other at that position.The oligonucleotide and the nucleic acid molecule are complemtary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotide units that can hydrogen bond witheach other.

[0077] Nucleic acid molecules and nucleotide sequences derived from thedisclosed molecules also can be defined as nucleotide sequences thathybridize under stringent conditions to the sequences disclosed, orfragments thereof.

[0078] “Specifically hybridizable” and “complementary” are terms whichindicate a sufficient degree of complementarity, such that stable andspecific binding occurs between an oligonucleotide and the targetnucleic acid. An oligonucleotide need not be 100% complementary to thetarget to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget molecule interferes with the normal function of the target andthere is a sufficient degree of complementarity to avoid non-specificbinding of the oligonucleotide to non-target sequences under conditionsin which specific binding is desired (for example, under physiologicalconditions in the case of in vivo assays) or under conditions in whichthe assays are performed.

[0079] Hybridization conditions resulting in particular degrees ofstringency will vary depending upon the nature of the hybridization,method of choice, and the composition and length of the hybridizingnucleic acid used. Generally, the temperature of hybridization and theionic strength (especially the Na⁺ concentration) of the hybridizationbuffer will determine the stringency of hybridization. Calculationsregarding hybridization conditions required for attaining particulardegrees of stringency are discussed in Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001).

[0080] Epitope.

[0081] A site on an antigen at which an antibody can bind, the moleculararrangement of the site determining the combining antibody. A portion ofan antigen molecule that determines its capacity to combine with thespecific combining site of its corresponding antibody in anantigen-antibody interaction.

[0082] Nucleotide Molecules That Hybridize.

[0083] Nucleotide molecules and sequences which are derived from thedisclosed nucleotide molecules as described above also can be defined asnucleotide sequences that hybridize under stringent conditions to thenucleotide sequences disclosed, or fragments thereof.

[0084] Genetic Fragment.

[0085] Any nucleic acid derived from a larger nucleic acid.

[0086] Heterologous.

[0087] Originating from a different organism or distinct tissue culture,such as from a different species or cell line.

[0088] Homologs.

[0089] Two nucleotide sequences that share a common ancestral sequenceand diverged when a species carrying that ancestral sequence split intotwo species.

[0090] Isolated.

[0091] An “isolated” biological component (such as a nucleic acid,polypeptide, protein, or organelle) has been substantially separated,produced apart from, or purified away from other biological components(for example, other chromosomal and extrachromosomal DNA and RNA, andpolypeptides) found in the cell of the organism in which the componentnaturally occurs. Nucleic acids, polypeptides, and proteins that havebeen “isolated” thus include nucleic acids and polypeptides purified bystandard purification methods. The term also embraces nucleic acids,polypeptides, and proteins that are chemically synthesized or preparedby recombinant expression in a host cell.

[0092] Nucleic Acid.

[0093] A deoxyribonucleotide or ribonucleotide polymer in either singleor double stranded form. Unless otherwise limited, this term encompassesknown analogues of natural nucleotides that hybridize to nucleic acidsin a manner similar to naturally occurring nucleotides. An“oligonucleotide” (or “oligo”) is a linear nucleic acid of up to about250 nucleotide bases in length. For example, a polynucleotide (such asDNA or RNA) which is at least 5 nucleotides long, such as at least 15,50, 100, or even more than 200 nucleotides long.

[0094] Operably Linked.

[0095] A first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably linked nucleic acid sequences are contiguous. Wherenecessary to join two protein coding regions, the operably linkedsequences are in the same reading frame.

[0096] Expression Control Sequence.

[0097] A nucleic acid sequence that affects, modifies, or influencesexpression of a second nucleic acid sequence. Promoters, operators,repressors, and enhancers are examples of expression control sequences.

[0098] ORF (Open Reading Frame).

[0099] A series of nucleotide triplets (codons) coding for amino acidswithout any termination codons. These sequences are usually translatableinto a peptide.

[0100] Ortholog.

[0101] Two nucleotide sequences are orthologs of each other if theyshare a common ancestral sequence and diverged when a species carryingthat ancestral sequence split into two species. Orthologous sequencesare also homologous sequences.

[0102] Parenteral.

[0103] Administered outside of the intestine and not via the alimentarytract. Generally, parenteral formulations are those that will beadministered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, or subcutaneously,and various surface applications including intranasal, intradermal, andtopical application, for instance.

[0104] Polypeptide.

[0105] Any chain of amino acids, regardless of length orpost-translational modification (for example, glycosylation orphosphorylation).

[0106] Polypeptide Sequence Homology.

[0107] In certain embodiments, a polypeptide is at least about 70%homologous to a corresponding sequence (such as SEQ ID NO:1) or a nativepolypeptide (such as HA), such as at least about 80% homologous, andeven at least about 95% homologous. Such homology is considered to be“substantial homology.”

[0108] Polypeptide homology is typically analyzed using sequenceanalysis software, such as the programs available from the GeneticsComputer Group (Madison, Wis., see the Genetics Computer Group website)

[0109] Portion of a Nucleic Acid Sequence.

[0110] At least 10, 20, 30, 40, 50, 60, 70, 80, or more contiguousnucleotides of the relevant sequence.

[0111] Promoter.

[0112] A promoter is one type of expression control sequence composedfrom an array of nucleic acid sequences that directs transcription of anucleic acid. A promoter includes necessary nucleic acid sequences nearthe start site of transcription, such as a TATA element. A promoter alsocan include distal enhancer or repressor elements that can be located asmuch as several thousand base pairs from the start site oftranscription. A promoter can be constitutive or inducible. An induciblepromoter directs transcription of a nucleic acid operably coupled to itonly under certain environmental conditions, such as in the presence ofmetal ions or above a certain temperature.

[0113] Protein Purification.

[0114] Polypeptides can be purified by any method known to one of skillin the art. Exemplary, non-limiting methods are described in: Guide toProtein Purification: Methods Enzymologyl, ed. Deutscher, AcademicPress, San Diego, 1997; and Scopes, Protein Purification: Principles andPractice, 3^(rd) ed., Springer Verlag, New York, 1994.

[0115] Purified.

[0116] The term purified does not require absolute purity; rather, it isintended as a relative term. Thus, for example, a purified nucleic acidis one in which the nucleic acid is more enriched than the nucleic acidis in its natural environment within a cell. In one embodiment, apreparation is purified if a component, such as a nucleic acid,represents at least 50% of the total amount of that component (e.g. thenucleic acid content) of the preparation.

[0117] Recombinant.

[0118] A recombinant nucleic acid is one that has a sequence that is notnaturally occurring, or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination can be accomplished by chemical synthesis orartificial manipulation of isolated segments of nucleic acids, forexample by genetic engineering techniques. Similarly, a recombinantprotein is one encoded for by a recombinant nucleic acid molecule. Theterm recombinant includes nucleic acids that have been altered solely bydeletion of a portion of the nucleic acid.

[0119] Resistance to Infection.

[0120] Animnals resistant to infection will demonstrate decreasedsymptoms of infection compared to non-resistant animals. Evidence ofresistance to infection can appear as, for example, lower rates ofmortality; increased life-spans measured after exposure to the infectiveagent; fewer or less intense physiological symptoms, such as fewerlesions; or decreased cellular or tissue concentrations of the infectiveagent. In one embodiment, resistance to infection is demonstrated by aheightened immune response.

[0121] Sequence Identity.

[0122] The similarity between two nucleic acid sequences, or two aminoacid sequences, is expressed in terms of the similarity between thesequences, otherwise referred to as sequence identity. Sequence identityis frequently measured in terms of percentage identity (or similarity orhomlogy); the higher the percentage, the more similar are the twosequences.

[0123] Methods of alignment of sequences for comparison are well-knownin the art. Various programs and alignment algorithms are described in:Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch,J. Mol. Bio. 48:443, 1970; Pearson and Lipman, Methods in Molec. Biology24: 307-331, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-90, 1988; Huang et al., Computer Applications in BioSciences8:155-65,1992; and Pearson et al., Methods in Molecular Biology24:307-31,1994. Altschul et al. (1994) presents a detailed considerationof sequence alignment methods and homology calculations.

[0124] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biological Information (NBCI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blasm, blastx, tblastn and tblastx.It can be accessed at the NCBI website.

[0125] Homologs of the nucleic acids and polypeptides described hereinare typically characterized by possession of at least 70% sequenceidentity counted over the full length alignment with a disclosedsequence using the NCBI Blast 2.0, gapped blastp set to defaultparameters. Such homologous nucleic acids or peptides will possess atleast 70%, at least 80%, or even at least 90% or 95% sequence identitydetermined by this method. When less than the entire sequence is beingcompared for sequence identity, homologs will possess at least 70%, suchas at least 85%, or even at least 90% or 95% sequence identity overshort windows of 10-20 amino acids. Methods for determining sequenceidentity over such short windows are described at the NCBI website.These sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs or other variantscould be obtained that fall outside of the ranges provided.

[0126] In addition to the peptide homologs described above, nucleic acidmolecules that encode such homologs are encompassed by alternativeembodiments. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide which the first nucleicacid encodes is immunologically cross reactive with the polypeptideencoded by the second nucleic acid. Another indication that two nucleicacid sequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions. Stringentconditions, as described above, are sequence dependent and are differentunder different environmental parameters.

[0127] Nucleic acid sequences that do not show a high degree of identitycan nevertheless encode similar amino acid sequences, due to thedegeneracy of the genetic code. It is understood that changes in nucleicacid sequence can be made using this degeneracy to produce multiplenucleic acid sequence that all encode substantially the samepolypeptide.

[0128] Nucleic cid molecules demonstrating substantial similarity may beof different types. A DNA molecule can demonstrate some degree ofidentity to an RNA molecule by comparing the sequences, where a Tresidue on the DNA molecule is considered identical to a U residue onthe RNA molecule.

[0129] Substantially Similar.

[0130] When optimally aligned (with appropriate nucleotide insertions ordeletions) with the other nucleic acid (or its complementary strand),there is nucleotide sequence identity in at least about 50%, 60%, 70%,80% or 90 to 95% of the nucleotide bases.

[0131] Therapeutic Agent.

[0132] Includes treating agents, prophylactic agents, and replacementagents made from nucleic acid and/or amino acid compositions describedherein.

[0133] Therapeutically Effective Amount or Effective Amount.

[0134] A quantity sufficient to achieve a desired effect in situ, invitro, in vivo, or within a subject being treated. For instance, theeffective amount can be the amount necessary to inhibit viralproliferation or to measurably alter progression of disease. In general,this amount will be sufficient to measurably inhibit virus (ISAV)replication or infectivity.

[0135] An effective amount can be administered in a single dose, or inseveral doses, for example daily, during a course of treatment. However,the effective amount can depend on the composition applied oradministered, the subject being treated, the severity and type of theaffliction, and the manner of administration.

[0136] The compositions disclosed have application in various settings,such as aquaculture, environmental containment, or veterinary settings.Therefore, the general term “subject being treated” is understood toinclude all fish that are or may be infected with a virus or otherdisease-causing microorganism that is susceptible to neutralization bythe compositions described herein.

[0137] Transduced, Transformed, and Transfected.

[0138] A virus or vector “transduces” a cell when it transfers nucleicacid into the cell. A cell is “transformed” by a nucleic acid transducedinto the cell when the DNA becomes stably replicated by the cell, eitherby incorporation of the nucleic acid into the cellular genome, or byepisomal replication. Transfection is the uptake by eukaryotic cells ofa nucleic acid from the local environment and can be considered theeukaryotic counterpart to bacterial transformation.

[0139] As used herein, the term transformation encompasses alltechniques by which a nucleic acid molecule might be introduced into acell.

[0140] Transgene.

[0141] An exogenous gene supplied by a vector.

[0142] Transgenic.

[0143] Of, pertaining to, or containing a gene, ORF, or other nucleicacid native to another species, microorganism, or virus. The term“transgenic” includes transient and permanent transformation, where thenucleic acid integrates into chromosomal DNA, including the germ line,or is maintained extrachromosomally.

[0144] Variants of Amino Acid and Nucleic Acid Sequences.

[0145] The production of proteins disclosed herein (for example, HA) canbe accomplished in a variety of ways. DNA sequences which encode for theprotein, or a fragment of the protein, can be engineered such that theyallow the protein to be expressed in eukaryotic cells, bacteria,insects, and/or plants. In order to accomplish this expression, the DNAsequence can be altered and operably linked to other regulatorysequences. The final product, which contains the regulatory sequencesand the nucleic acid, is referred to as a vector. This vector can thenbe introduced into the eukaryotic cells, bacteria, insect, and/or plant.Once inside the cell, the vector allows the protein to be produced.

[0146] The DNA can be altered in numerous ways without affecting thebiological activity of the encoded protein. For example, PCR can be usedto produce variations in the DNA sequence which encodes an ISAV peptide.Such variants can be variants that are optimized for codon preference ina host cell that is to be used to express the protein, or other sequencechanges that facilitate expression.

[0147] At least two types of cDNA sequence variant can be produced. Inthe first type, the variation in the cDNA sequence is not manifested asa change in the amino acid sequence of the encoded polypeptide. Thesesilent variations are simply a reflection of the degeneracy of thegenetic code. In the second type, the cDNA sequence variation doesresult in a change in the amino acid sequence of the encoded protein. Insuch cases, the variant cDNA sequence produces a variant polypeptidesequence. In order to preserve the functional and immunologic identityof the encoded polypeptide, certain embodiments utilize amino acidsubstitutions that are conservative.

[0148] Variations in the cDNA sequence that result in amino acidchanges, whether conservative or not, can be minimized in order topreserve the functional and immunologic identity of the encoded protein.Variant amino acid sequences can, for example, be 70, 80%, 90%, or even95% identical to the native amino acid sequence.

[0149] Vector.

[0150] A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements. Avector can transduce, transform or transfect a cell, thereby causing thecell to express nucleic acids and/or proteins other than those native tothe cell. A vector optionally includes materials to aid in achievingentry of the nucleic acid into the cell, such as a viral particle,liposome, protein coating, or the like. Plasmids are often used asvectors to transform fish cells.

[0151] ISAV Specific Nucleic Acids and Polypeptides

[0152] Polypeptides and nucleic acid molecules are disclosed herein, asare and treatments for protecting fish, shellfish, and otheraquacultured organisms against ISAV. The nucleic acids include segmentsof the ISAV genome, such as the segments described herein and summarizedin Table 5 below, or fragments thereof. Also included are fragments ofthe ISAV genome that overlap the individual segments summarized in Table5.

[0153] ISAV polypeptides are described herein, as are nucleic acids thatencode the ISAV polypeptides. ISAV polypeptides include, but are notlimited to, P1, PB1, (nucleotprotein) NP, P2, P3, hemaglutinin (HA), P4,P5, P6, and P7. Thus, polypeptides having a sequence as set forth as SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO:18, an antigenic fragment thereof, or a conservative variant thereof,are provided herein.

[0154] Polypeptides can be divided into sections, such as an N-terminaland a C-terminal portion. Thus, in one embodiment, polypeptide fragmentsare provided that include the N-terminal or the C-terminal portion of anISAV polypeptide.

[0155] Antigenic fragments of an ISAV polypeptide are provided herein.An antigenic fragment is any ISAV polypeptide that can produce an immuneresponse in fish. The immune response can be a B cell or a T cellresponse, or induction of a cytokine.

[0156] Also provided herein are nucleic acids that encode and ISAVpolypeptide. In one embodiment, a nucleic acid is provided that encodesa P1 polypeptide. One specific non-limiting example of a P1 polypeptideis the sequence set forth as SEQ ID NO:2, a fragment, or a conservativevariant thereof.

[0157] In another embodiment, a nucleic acid is provided that encodes ahemaglutinin (HA) polypeptide. One specific, non-limiting example of anHA polypeptide is the sequence as set forth as SEQ ID NO:12, a fragment,or a conservative variant thereof.

[0158] In a further embodiment, a nucleic acid is provided that encodesa PB1 polypeptide. One specific, non-limiting example of an PB1polypeptide is the sequence as set forth as SEQ ID NO:4, a fragment, ora conservative variant thereof.

[0159] Nucleic acids are also disclosed herein that are substantiallysimilar to particular segments, such as nucleic acids that are at least70% identical to SEQ ID NO: 1, at least 85% identical to SEQ ID NO: 3,or at least 85% identical to SEQ ID NO: 11. Thus, in one embodiment, anucleic acids is provided that is are at least 75% identical to SEQ IDNO: 1, at least at least 80% identical to SEQ ID NO: 1, at least 85%identical to SEQ ID NO: 1, at least 90% identical to SEQ ID NO: 1, or atleast 95% identical to SEQ ID NO: 1. In another embodiment, a nucleicacid is provided that is at least 90% identical to SEQ ID NO: 3, atleast 95% identical to SEQ ID NO: 3, or at least 99% identical to SEQ IDNO:3. In a further embodiment, a nucleic acid is provided that is atleast 90% identical to SEQ ID NO: 11, at least 95% identical to SEQ IDNO: 11, or at least 99% identical to SEQ ID NO:11.

[0160] In yet another embodiment, nucleic acids are provided thatconsist essentially of an ISAV nucleic acid sequences, such as a nucleicacid having a sequence as set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 16.

[0161] The nucleic acids disclosed herein can be operably linked to aheterologous nucleic acid, such as an expression control sequence. Inone embodiment, the expression control sequence is a promoter, such asan interferon response element, beta-actin, a cytokine promoter, acytomegalovirus promoter, or a fish viral promoter. In particularembodiments, the promoter is an inducible promoter, such as a heat shockpromoter, or a promoter induced by a hormone or a metal ion. Nucleicacid compositions can contain other elements, such as additionalexpression control elements, structural sequences, origins ofreplication, or multiple coding sequences. In particular embodiments, anexpression control sequence operably linked to a nucleic acid encodingan ISAV polypeptide is included in a vector, including, but not limitedto, a plasmid, a viral vector, a phagemid, or a cosmid. Cloning vectorsinclude, but are not limited to, those described in U.S. Pat. No.5,998,697. Viral vectors include, but are not limited to, retroviral oradenoviral vectors.

[0162] The nucleic acid compositions described herein can be utilized invitro, in vivo, or in situ. For example, a nucleic acid at least 70%identical to SEQ ID NO: 1 could be used to study an antigenic epitope ofinterest for in vitro production and manipulation, or to study itseffect on cell physiology or activity in vivo, or for tissue-specificexpression analysis in situ. Particular uses of these nucleic acidcompositions also are illustrated in the Examples below.

[0163] In some embodiments, the nucleic acid molecule encodes anantigenic sequence, such as an antigenic sequence for pathogens ofaquacultural animals. Aquacultural animals include fish (both bony andcartilaginous fish), shellfish and other arthropods, and molluscs.Particular exemplary aquicultural animals include, but are not limited,to the following: salmonids, such as rainbow trout (Oncorhynchusmykiss), coho salmon (O. kisutch), chinook salmon (O. tshawytcha), amagosalmon (O. rhodurus), chum salmon (O. keta Walbaum), sockeye salmon (O.nerka), Atlantic salmon (Salmo salar), arctic char (Salvelinus alpinus),brown trout (Salmo trutta), cutthroat trout (Salmo clarkii), and brooktrout (Salvelinus fontinalis); catfish (Ictalurus punctatus); tilapia(Oreochromis niloticusand and Oreochromis mozambicus); sea bream(Archosargus rhomboidalis), seabass (Dicentrarchus labrax); flounder(Paralichthys dentatus); sturgeon (Scaphirhynchus albus); eels(including members of the order Anguilliformes, class Actinopterygii,such as Conger spp., Ariosoma spp., Gnathophis spp., Coloconger spp.,Anguilla spp., Nessorhamphus spp., Cynoponticus spp., Anarchias spp.,Echidna spp., Enchelycore spp., Gymnothorax spp., and Uropterygiusspp.); cephalopods (octopi and squids); crustaceans (including lobsters,prawns, shrimp, crabs, and crayfish in the order Decapoda); and bivalves(clams and oysters, such as Ostrea edulis and Pisidium spp.). In someembodiments, the aquaculture animal is a fish, such as a salmonid. Inparticular embodiments, In some embodiments, the nucleic acid moleculeencodes a polypeptide that is an antigenic sequence, such that uponintroduction in fish, an immune response is induced against ISAV.

[0164] Eliciting an Immune Response in Fish

[0165] Some embodiments employ nucleic acid compositions containingnucleic acid sequences encoding antigenic epitopes. In such embodiments,the nucleic acid composition includes an expression control sequenceoperably linked to a nucleic acid sequence encoding an antigenicepitope, thus driving expression of the nucleic acid sequence andeliciting an immune response to the antigenic epitope in the fish. Inparticular embodiments, the antigen expressed is a polypeptide encodedby ISAV, which elicits an immune response in the fish against ISAV.

[0166] In any such embodiment, the fish utilized can belong to aparticular species, such as rainbow trout, coho salmon, chinook salmon,amago salmon, chum salmon, sockeye salmon, Atlantic salmon, arctic char,brown trout, cutthroat trout, brook trout, catfish, tilapia, sea bream,seabass, flounder, and sturgeon.

[0167] Exemplary, non-limiting uses of these nucleic acid compositionsare described in the Examples below.

[0168] In certain embodiments, any of the nucleic acid compositionsdescribed herein is used to transform fish tissue to produce atransgenic fish. In such embodiments, a nucleated cell of the transgenicfish is transformed with a nucleic acid sequence substantially similarto the nucleic acid sequences described herein (for example, SEQ IDNOS.: 1, 3, 5, 7, 9, 11, 13, and 16). In particular embodiments, thenucleic acid is at least 70% identical to SEQ ID NO: 1, at least 85%identical to SEQ ID NO: 3, or at least 85% identical to SEQ ID NO: 11,operably linked to a heterologous nucleic acid sequence.

[0169] If it encodes an antigenic epitope, expression of the nucleicacid sequence can induce an immune response to the antigenic epitopewithin the fish or other aquaculture animal. In such embodiments, theanimal exhibits an increased resistence to infection by ISAV as comparedto a non-transformed animal of the same species.

[0170] In alternative embodiments, the animal subject is treated with apolypeptide composition that functions as an antigenic epitope andinduces an immune response within that subject, such as the polypeptidesa sequence as set forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 17, or SEQ ID NO: 18, an antigenic fragment thereof, or aconservative variant thereof. In some embodiments, the antigenicpolypeptide is a fusion protein, such as a polypeptide as set forth inSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ IDNO: 18, an antigenic fragment thereof, or a conservative variantthereof, coupled to a fusion partner. Such fusion partners include, butare not limited to, antigens from other fish viruses (for example,glycoprotein from rhabdovirus, birnavirus, reovirus, nodavirus, herpesvirus, or infectious pancreatic necrosis virus), ISS DNA elements, orT-cell epitopes.

[0171] The antigenic polypeptides can be obtained by recombinantmethods, such as expression in eukaryotic or bacterial cell culture, orcan be chemically synthesized. In particular embodiments, the antigenicpolypeptides are recombinantly expressed in a non-mammalian eukaryoticcell culture, such as a fish cell culture, for example a CHSE-214, TO,SHK, RTG-2, or EPC cell culture. Thus, an antigenic polypeptide can beprepared by transforming fish cells with a nucleic acid vector encodingan antigenic polypeptide (including one that is a fusion protein), asdescribed above, culturing the host cells under conditions suitable forexpressing the antigenic polypeptide, and then recovering the antigenicpolypeptide from the cell culture. Additionally, such cell cultures canbe transformed with multiple nucleic acid vectors, thus expressingmultiple antigenic polypeptides.

[0172] Recovered antigenic polypeptides can then be purified and readiedfor delivery to the subject (as described above), and the antigenicpolypeptide can be combined with a pharmaceutically acceptable salt,carrier, adjuvant, or diluent, and/or other active or inactiveingredients, to form a pharmaceutical composition. The amount orconcentration of the antigenic polypeptide within the pharmaceuticalcomposition can vary according to factors such as the effectiveness ofthe antigenic polypeptide in inducing an immune response within thespecies of the subject, the severity of the disease or condition to betreated, the route or frequency of administration, or other relevantfactors. These compositions also can be tested for immunogenicity priorto delivery to a subject using an in vitro assay, such as one of theassays described in the Examples below.

[0173] Once prepared, an effective amount of the antigenic polypeptideor pharmaceutical composition is delivered to the subject via a suitableroute of administration, for example, intramuscular, intraperitoneal,oral, immersion, or ultrasound administration. An effective amount isany amount that enhances the immunocompetence of the subject treated andelicits some immunity against ISAV, for example, by delaying,inhibiting, or even preventing the onset or progression of ISA. In someembodiments, the subject's immune system is stimulated by at least about15%, such as by at least about 50%, or even at least about 90%.

EXAMPLES

[0174] The following examples are intended to illustrate the invention,but not to limit it in any manner, either explicitly or implicitly.While these examples are typical of those that might be used, otherprocedures, methodologies, or techniques known to those skilled in theart alternatively can be used.

Example 1 Vaccine Trial to Test the Efficacy of the Whole Killed ISAVVaccine

[0175] Atlantic salmon, each weighing about 90 g, were anaesthetized andintraperitoneal injected with 0.2 ml of a solution of whole killed ISAV.Four groups of twenty fish in each group were studied; two groups wereinjected with whole killed ISAV and two groups were injected with anequivalent amount of saline.

[0176] Following vaccination, the salmon were acclimated to saltwater at12° C. and held for 798 degree days prior to challenge with ISAV.Twenty-four native Atlantic salmon were anaesthetized, fin clipped foridentification, and intraperitoneal injected with 1 ml ISAV infectedCHSE-214 cell culture supernatant (1×10⁷ TCID₅₀/ml). Six of these fishwere added to each of the four tanks containing the groups of either thevaccinated or control fish. Saline-injected control salmon experienced acumulative mortality of 57.5% when challenged with ISAV by cohabitation.Vaccinated salmon had a cumulative mortality of 17.5%. The RPS value ofthe ISAV whole killed vaccine was 70.0%.

Example 2 Humoral Immune Response to Whole Killed ISAV

[0177] Atlantic salmon were vaccinated with the two different serials ofwhole killed ISAV in MV4 at two different antigen doses, and sera wascollected at 350 degree-days, 696 degree-days, and 972 degree-days aftervaccination.

[0178] ISAV-specific antibodies in the sera were detected byenzyme-linked immunosorbent assay (ELISA). ISAV antigen was dried ontowells of an ELISA plate overnight at 37° C. Wells were rinsed threetimes with PBS/Tween and, serial dilutions of anti-ISAV Atlantic salmonsera were added in triplicate. After one hour, wells were rinsed threetimes with PBS/Tween. A second antibody, mouse anti-salmonimmunoglobulin, was added to each well, the plates were incubated forone hour, and then rinsed with PBS/Tween in triplicate. After incubationwith a third antibody, goat anti-mouse IgG conjugated to alkalinephosphatase, the wells were washed with PBS/Tween and developercontaining p-nitrophenyl phosphate was added. The absorbance wasmeasured at 405 nm.

[0179]FIG. 3 illustrates the results of this humoral response trial.Antibody levels were reported as a percent of the value obtained withmAb 10A3 to normalize the variation between ELISA plates. A 1× dose ofthe whole killed ISAV vaccine serial 327 elicits a humoral immuneresponse at 972 degree-days post-vaccination.

Example 3 Virus and RNA Purification

[0180] Virus was prepared by inoculating CHSE-214 cell monolayers in a6300 cm² Cell Factory® (Nalge Nunc International, Rochester, N.Y., USA)with ISA virus. Following complete cell lysis, the cell culturesupernatant was harvested from the Cell Factory® as disclosed by themanufacturer and filtered through a sterile 0.45 micron filter to removeextraneous cell debris. After dialysis against solid polyethylene glycolto reduce the volume, the cell culture supernatant was centrifuged fortwo hours at 24,000 rpm using a SW28 rotor and a Beckman L8-70Multracentrifuge (Beckman Coulter, Inc., Fullerton, Calif., USA). Thepelleted virus was resuspended in TNE, layered on a 25, 35 and 45%sucrose gradient and centrifuged for 3 hours at 27,000 rpm using a SW28rotor and a Beckman L8-70M ultracentrifuge. Virus at the interface ofthe 35 and 45% sucrose layers was collected, resuspended in TNE andcentriged for two hours at 24,000 rpm using a SW28 rotor and a BeckmanL8-70M ultracentrifuge. The fraction collected from the 35-45% interfacewas enriched with material that reacted with an ISAV-specific monoclonalantibody. Viral RNA was isolated from the pelleted virus using Trizol(Gibco) as described by the manufacturer and then used to construct cDNAlibraries.

[0181] Purified ISAV was resuspended in SDS-sample buffer. Thesolubilized proteins were separated by SDS-PAGE on a 5% stacking gel anda 12% resolving gel and visualized by Coomassie blue staining. As shownin FIG. 2, after SDS-PAGE, four distinct protein bands were evident: 72kDa, 47 kDa, 42 kDa, 25 kDa.

[0182] Seven proteins from purified ISAV were subjected to N-terminalamino acid sequence analysis. The proteins of purified ISAV wereseparated by SDS-PAGE, blotted onto PVDF membrane (BioRad Laboratories,Hercules, Calif.) and stained with 0.1% Coomassie blue R-250 in 40%methanol/1% acetic acid. The stained protein bands were cut out of themembrane and subjected to N-terminal amino acid sequence analysis usingan Applied Biosystems model 470A gas-phase sequencer (AppliedBiosystems, Inc., Foster City, Calif.) or an Applied Biosystems model473 liquid-phase sequencer with on-line phenylthiohydantoin analysis.The results of this sequencing analysis are shown in Table 2. TABLE 2N-terminal amino acid sequence analysis of ISAV proteins Protein MWSimilarity (kDa) Sequence analysis analysis 25 KVSFDMA; SLQGPVA(internal No similarity found sequence) 35 N-terminally blocked N/A 38N-terminally blocked N/A 40 RLXLRNHPDTTWIGDSRSDQSRXNQ Putative segment(N-terminal sequence) 7 ISAV; segment 4 Influenza C 42RLXLRNHPDTTWIGDSRSDQSRXNQ HA (segment 6) (N-terminal sequence) 47EPXIXENPTXLAI (N-terminal sequence) 5: E-7 (segment 5) 72 N-terminallyblocked N/A

Example 4 Construction of cDNA Libraries

[0183] Strategies for Cloning the ISA V Genome.

[0184] Approach 1: First strand cDNA was synthesized from ISA vRNA byreverse transcription with the ISAV-specific primer (SEQ ID NO: 19):

[0185] 5′-AAGCAGTGGTAACAACGCAGAGTAGCAAAGA-3′

[0186] RNA (100 ng) isolated from purified ISAV or CHSE-214 cells(control) was mixed with ISAV primer (20 pmol/μl), incubated at 80° C.for 5 min and then combined with the following in a total of 20 μl: 4 μA5× first strand buffer (Gibco Invitrogen Corp., Carlsbad, Calif.), 2 μl10 mM dNTP mix (Boehringer Mannheim), 1 μl 0.1 M DTT (Gibco) and 1 μlSuperscript II reverse transcriptase (15 U/μl; Gibco). The mixture wasincubated at 25° C. for 10 min and then at 42° C. for 1 hr.

[0187] The first strand ISAV cDNA products synthesized by reversetranscription were PCR amplified using the ISAV primer and randomhexamers. To the first strand reaction, the following components wereadded in a total of 100 μl: 1.5 μl 10 mM DNTP mix (Boehringer Mannheim,1.25 μl ISAV primer (20 pmol/μl), 1 μl random hexamers (25 pmol/μl;Gibco), 10 μl 10× PCR buffer with Mg²⁺ (Boehringer Mannheim), 1 μl Taq(5 U/μl; Boehringer Mannheim). After 35 cycles of 94° C. for 30 sec, 59°C. for 45 sec and 72° C. for 1 min, the PCR products were extended for10 min at 72° C. The amplified cDNA products were separated by agarosegel electrophoresis, gel purified and then cloned into the pGEM-T vectoras described by the manufacturer (Promega).

[0188] Approach 2: First strand cDNA was synthesized from ISA vRNA byreverse transcription with random hexamer primers. RNA (100 ng) isolatedfrom purified ISAV or CHSE-214 cells (control) was mixed with randomhexamers (50 ng/μl; Gibco), incubated at 65° C. for 5 min, placed on icefor 2 min and then combined with the following in a total of 20 μl: 4 μl5× first, strand buffer (Gibco), 2 μl 10 mM dNTP mix (BoehringerMannheim), 1 μl 0.1 M DTT (Gibco) and 1 μl Superscript II reversetranscriptase (15 U/μl; Gibco). The mixture was incubated at 25° C. for10 min and then at 50° C. for 50 min.

[0189] The TimeSaver cDNA synthesis kit (Pharmacia) was used for secondstrand cDNA synthesis. The first strand reaction was added to the secondstrand reaction mix, incubated at 12° C. for 30 min and then at 22° C.for 1 hr. After spin column purification, the blunt ended, doublestranded cDNAs were cloned into dephosphorylated, SmaI digested pUC18(Pharmacia) as outlined by the manufacturer.

[0190] For both libraries, E. coli DH5α (Gibco) was transformed with theligation reactions and the ampicillin-resistant colonies containingeither pGEM-T or pUC18 with cloned ISAV cDNA were selected by blue/whitescreening. The white colonies were transferred to 96 well platescontaining 200 μl LB/ampicillin (250 μg/ml)/15% glycerol per well, grownovernight at 37° C. and stored at −20° C.

[0191] RT-PCR Amplification of Segments 2, 6 and 8 from ISA V CCBB.

[0192] First strand cDNAs for segments 2, 6 and 8 were synthesized fromISA virus RNA by reverse transcription using primers outlined in Table 3and conditions described above. PCR amplification was used for secondstrand cDNA synthesis; after 30 cycles of 95° C. for 1 min, 50° C. for 1min and 72° C. for 2 min, the PCR products were extended for 10 min at72° C. (see Table 3 for primers). RT-PCR products were gel purified asdescribed by the manufacturer (Qiagen). Table 3. RT and PCRoligonucleotide DNA primers for RNA segments 2, 6 and 8 of ISA virusisolate CCBB. !Segment? Primer name? Primer sequence (5′-3′) 2 seg2-5′F-mRNA GAACGCTCTTTAATAACCATG seg 2-3′R-mRNA TCAAACATGCTTTTTCTTC 6 HAforward AGCAAAGATGGCACGATTC HA reverse TGCACTTTTCTGTAAACGTACAAC 8 seg8-5′F-mRNA AAGCAGTGGTAACAACGCAGAGTCTATCTACCATG seg 8-3′R-mRNATTATTGTACAGAGTCTTCC

[0193] Selection and Identification of ISAV Clones from the cDNALibraries.

[0194] The contents of one 96-well plate were transferred to one HybondN⁺ membrane (Amersham) then placed on top of an LB agar plate containingampicillin (250 μg/ml). Clones were grown on the filters at 37° C.overnight and the filters were processed on soaking pads saturated withthe following solutions: 0.5 N NaOH (7 min); 1 M Tris-HCl pH 7.4 (2min); 1 M Tris-HCl pH 7.4 (2 min); 0.5 M Tris-HCl pH 7.4, 1.5 M NaCl (4min). The filters were transferred to a bath of 2×SSC (1×SSC is 0.15 MNaCl, 0.015 M Na₃ citrate), 1% sodium dodecyl sulfate (SDS) and thensoaked in 2×SSC. After a brief wash in chloroform, the filters were airdried and then baked at 80° C. for 2 hrs. Prehybridization of thefilters for 2 hr in 6×SSC, 0.5% SDS, 5× Denhardt's and 0.1 mg/ml E. colitRNA (Sigma) was followed by hybridization with a probe labelled with[α³²P] dCTP by nick translation. Nick translation was done as outlinedby the manufacturer (Amersham). The libraries were initially screenedusing gel purified, RT-PCR amplified cDNA for segments 2, 6 or 8 of ISAVisolate CCBB. The remaining segments were identified using probesconsisting of gel purified, restriction enzyme fragments digested fromthe plasmids of randomly selected library clones. Library clones weregrouped based on the probe to which they hybridized (see Table 4). Eightdistinct cDNA hybridization groups were identified. Of these, two groupswere found in cDNA library 1 and all but one segment of the ISAV genomein cDNA library 2 (see Table 4).

[0195] Plasmid DNA isolated from representative clones of each groupusing Qiaprep columns (Qiagen) was sequenced at the University of MaineCore Sequencing Facility. Only those sequences that matched otherorthomyxovirus sequences or that did not match non-viral sequences wereanalyzed further. TABLE 4 Summary of groups formed from screening ISAvirus cDNA libraries. Number of positive clones Origin of cDNA librarycDNA library Probe probe approach 1¹ approach 2² 5: E-6 approach 2 0 33Segment 2 RT-PCR 0 41 1-1#2; 5-1#1 approach 1 212; 1144 6; 43 2: C-5; 4:D-8 approach 2 0 5; 38 5: E-7 approach 2 0 14 Segment 6 RT-PCR 0 0 2:B-10 approach 2 0 50 Segment 8 RT-PCR 6 10

[0196] Northern Blot Hybridization.

[0197] Northern blot analysis was used to correlate each representativesequence with a specific ISAV genomic segment. Total RNA was isolatedfrom CHSE-214 cell monolayers or CHSE-214 cell monolayers infected withISAV using Trizol (Gibco) as outlined by the manufacturer. The RNA wasseparated on a 2% agarose gel containing formaldehyde and transferredonto Hybond N⁺ membrane (Amersham) in 10×SSC by capillary action asdescribed in Fourney et al., Focus, 10:5-7 (1992).

[0198] The probes used for Northern blot analysis were gel purified,restriction enzyme fragments digested from the plasmids of appropriatecDNA library clones. The probes were labelled with [α³²P]dCTP (NEN) bynick translation (Amersham) and hybridized to the blots at 42° C. for 18hr in ULTRAhyb™ (Ambion). The membranes were washed 2×5 min in2×SSC-0.1% SDS at 42° C. and then 2×15 min in 0.1×SSC-0.1% SDS at 42° C.The results were recorded on Kodak X-OMAT AR film.

[0199] The probes used in the Northern blot hybridization experimentswere derived from four clones constructed using approach 2, one cloneconstructed using approach 1 and RT-PCR products of the three knownsegments (see Table 3). A single RNA blot was consecutively probed witheach of the eight individual probes. One probe was hybridized to theNorthern blot and the results were visualized by autoradiography. Thenext probe was hybridized to the same Northern blot, the results werevisualized and compared with the results from the previoushybridization. By repeating this process with each of the eight probes,each individual probe and its corresponding nucleotide sequence wascorrelated with a specific RNA segment.

[0200] Eight RNA segments were identified; segments 1 and 2 were bothapproximately 2400 nucleotides in length. The ISAV RNA segmentcorresponding to each cDNA clone is summarized in Table 5. The genomesegments are numbered with respect to their mobility in agarose gels,from the slowest to the fastest and comprise a genome of 14,500nucleotides. TABLE 5 RNA segments of ISAV isolate CCBB, their genes andencoded proteins Molecular Length of Nascent weight segment¹ Length ofEncoded polypeptide predicted Segment Clone (kb) ORF (bp) protein length(aa) (kDa) 1 5:E-6 2.4 1749 P1 — — 2 PB1 2.4 2127 PB1 709 80.5 31-1#2/5-5#1 2.2 1851 NP 617 68.0 4 2:C-5/4:D-8 1.9 1737 P2 579 65.3 55:E-7 1.6 1335 P3 445 48.8 6 HA 1.5 1185 HA 395 43.1 7 2:B-10 1.3 771 P4257 28.6 441 P5 147 16.3 8 NS 1.0 705 P6 235 26.5 552 P7 184 20.3

[0201] Purified cellular RNA was separated on a 2% agarose gel andtransferred to a Hybond N+ membrane. Lanes 1-7 & 10 contain cellular RNAfrom CHSE cells infected with ISA virus isolate CCBB; lane 8 containscellular RNA from ISA virus isolate ME-01; lane 9 contains cellular RNAfrom CHSE cells infected with ISA virus isolate NB-99; and lane 11contains cellular RNA from naive CHSE cells.

[0202] The RNA blot was consecutively hybridized with radioactivelylabeled DNA probes specific for one of the ISA virus RNA segments. Theresults recorded by autoradiography after the addition of each singleprobe to the same RNA blot are shown in lanes 1-11. The probes areidentified by segment (according to Table 5 above): lane 1, segment 3;lane 2, segment 4; lane 3, segment 6; lane 4, segment 1; lane 5, segment5; lane 6, segment 7; lane 7, segment 8; lanes 8-11, segment 2.Molecular weight standards on the left are in kbp. The RNA segments arelabeled on the right.

[0203] Construction of Full-Length Clones of Each ISAV Genome Segment.

[0204] Full-length cDNA sequence for each of the ISAV RNA segments, withthe exception of segment 1, was generated by rapid amplification of cDNAends (RACE) PCR using the RLM-RACE kit (Ambion). The PCR products werecloned into either pCR®2.1-TOPO® or pGEM-T as directed by themanufacturers (Invitrogen or Promega, respectively) and then sequenced.AssemblyLIGN 1.0.9b (Oxford Molecular Group) was used to order theoverlapping sequenced DNA fragments for construction of the full-lengthsequence.

[0205] PCR primers were designed from the consensus sequence obtainedfor each ISAV RNA segment and used to amplify full-length cDNA sequencefor each segment with the exception of segment 1. The PCR product foreach segment was cloned into pGEM-T as directed by the manufacturer(Promega) and DNA from three representative clones was sequenced. Thecomputer programs contained in MacVector™ 6.5.3 (Oxford Molecular Group)were used to identify open reading frames and regions of localsimilarity. The nucleotide and predicted amino acid sequence for eachopen reading frame were analyzed by BLAST searches through the NationalCenter for Biotechnology Information server (Altschul et al., 1990;Pearson & Lipman, 1988) or the Influenza database (Los Alamos NationalLaboratory). The most likely cleavage sites for signal peptidase in HAand 5:E-7 were determined using SignalP V1.1 (Nielsen et al., 1997).

[0206] The length of each gene, the corresponding encoded polypeptide(s)and the predicted molecular weights of the translated proteins aresummarized in Table 1. Only partial sequence from segment 1 wasobtained. The cDNA sequence of segments 1-6 was predicted to encode oneopen reading frame. Segments 7 and 8 each were predicted to encode twoproteins.

[0207] Comparison of the cDNA nucleotide and predicted amino acidsequences for the ISA virus genome to those listed in the GenBank andInfluenza databases showed that RNA segments 1 and 5 of ISA virusisolate CCBB were unique. RNA segments 2, 3, 4 and 6 were found toencode the putative proteins PB1, NP, PA and HA, respectively. Thepredicted sequences of the P6 and P7 proteins encoded on RNA segment 8were similar to the sequences of the two open reading frames (orf) onsegment 8 from other ISA virus isolates.

[0208] The protein sequence of the partial open reading frame encoded onsegment 1 was unique. The predicted amino acid sequence of PB 1, encodedby RNA segment 2, was 82.2 to 84.5% similar to the amino acid sequencesof PB1 proteins from Norwegian (AJ002475) and Scottish (AF262392) ISAvirus isolates. The assignment of NP to the open reading frame encodedon RNA segment 3 was based on nucleotide sequence similarity to theinfluenza A NP RNA binding region (see FIG. 4) and to the putative NPsequence described by Snow & Cunningham (2001). The sequence for theCCBB ISA virus NP was highly conserved, sharing 96.6% identity to thatreported for the Scottish NP (AJ276858). The predicted protein sequenceof P2 from RNA segment 4 had 99% identity to the putative PA sequence(AF306548) described by Ritchie et al. (2001). The nucleotide sequencesfor segment 5 of the Scottish (AF429988), Norwegian (AF429987) and Maine(AF429986) isolates of ISAV were 76.4, 76.0 and 99.7% similar to thecorresponding sequence of ISAV isolate CCBB.

[0209] The predicted translation of the open reading frame encoded byRNA segment 6 shared 84.8 to 84.3% identity to the predicted HA proteinsequences for ISA virus isolates from Norway (AF302799) and Scotland(AJ276859), and 99.2% identity to the Maine ISA virus isolate(AY059402). The nucleotide sequence for ISAV CCBB segment 7 had 99.6%identity with a reported ISAV sequence (AX083264). The P4 and P5proteins encoded on segment 7 had 99.2 to 99.3% identity to thetranslations predicted for orf1 and orf2 from the reported sequence(AX083264). The nucleotide sequence for segment 8 of the Norwegian(AF429990) and ME/01 (AF429989) isolates of ISAV was 88.7-99.9%identical to the corresponding sequence from ISAV isolate CCBB. Ourresults confirmed that segment 8 encoded two proteins as previouslyreported by Mjaaland et al. (1997). The amino acid sequence translatedfrom the largest open reading frame was 75.6-97.9% identical to thesequence previously reported for Norwegian (AF262382), Scottish(AJ242016) and Canadian (AJ242016) isolates of ISA virus.

[0210]FIG. 4 shows the amino acid sequence alignment of the RNA bindingdomain of NP from influenza virus A and B with the putative NP RNAbinding domain from ISA virus as predicted using the Clustal W system.ISAV NP, aa 189-307 from accession number AF404345; Inf A NP, aa 90-188from accession number P15675; Inf B NP, aa 149-249 from accession numberP04666. Identical amino acids and amino acid residues with similarity inphysical and chemical properties are indicated as * and {circumflex over( )}, respectively. The NP RNA binding domain from influenza viruses Aand B was taken from Kobayashi et al. (1994).

Example 5 Humoral Immune Response

[0211] Anti-ISA virus antibodies were generated in Atlantic salmoninjected with tissue culture supernatant from ISA virus-infected CHSEcell monolayers. Anti-ISA virus antibodies were also generated inrainbow trout vaccinated with DNA vaccines expressing a ISAV-specificantigens. Mouse polyclonal and monoclonal antibodies (mAbs) to ISA viruswere generated by Rob Beecroft (Immuno-Precise Antibodies Ltd.).ISAV-specific immunoreactive antigens were detected by IFAT, ELISA,Western blot and serum neutralization assays.

[0212] Indirect Fluorescent Antibody Technique (IFAT)

[0213] IFATs were used to screen the ISAV-specific monoclonal antibodies(mAb). CHSE-214 cells infected with ISAV were fixed to a glass slidewith 100% acetone, blocked with 3% skim milk buffer, incubated withISAV-specific mAb 10A3, washed and reacted with TRITC-labelled goatanti-mouse antibody (Sigma). The slide was washed, air dried, and fixedto a glass slide with Cytoseal 60 (Stephens Scientific).

[0214] Viral-infected cells were stained a deep red whereas controlslides of naive CHSE cells were negative by IFAT with mAb 10A3.

[0215] ELISA

[0216] The levels of ISAV-specific antibodies in serum from Atlanticsalmon infected with ISAV or rainbow trout vaccinated with a DNA vaccinewere determined by enzyme-liked immunosorbent assay (ELISA). DNAvaccines tested were pISA-HA (NA), pISA-HA (Nor), pISA-seg7, andpISA-seg8.

[0217] ISAV antigen was dried onto wells of an ELISA plate overnight at37° C. Wells were rinsed three times with PBS/Tween and then serialdilutions of anti-ISAV sera were added in triplicate. After 1 hr, wellswere rinsed three times with PBS/Tween. The second antibody, mouseanti-salmon/rainbow trout immunoglobulin (Rob Beecroft), was added toeach well. The plates were incubated for 1 hr and then rinsed withPBS/Tween in triplicate. After incubation with the third antibody, goatanti-mouse IgG conjugated to alkaline phosphatase, the wells were washedwith PBS/Tween and developer containing p-nitrophenyl phosphate wasadded. The absorbance was measured at 405 nm.

[0218] ISAV-specific antibodies were detected in sera from fish injectedwith either live ISAV (FIGS. 4 and 5) or with DNA vaccines expressingISAV-specific antigens (FIG. 5).

[0219] Antibody studies are conducted in Atlantic salmon vaccinated withan ISAV DNA vaccine, an ISAV recombinant vaccine or a whole killed ISAVvaccine {DNA vaccines: pISA-NP, pISA-Ac, pISA-HA (NA), pISA-HA (Nor),pISA-seg7; recombinant vaccines: rHA-1; whole killed vaccines: 1×, 2×and 4× doses of formalin killed ISAV}. Sera samples are collected from 5fish/timepoint at 4, 6, 8, 10 and 12 weeks post-vaccination.

[0220]FIG. 5 shows the titration of ISAV-specific antibodies fromAtlantic salmon infected with ISAV. Fish 1 had not been exposed to ISAVand, thus, the serum was used as a negative control. Fish 45 wasinjected with ISAV, and the ELISA results indicated that thecorresponding serum contained ISAV-specific antibodies. Sera from fish 1and fish 45 were negative when tested by ELISA using plates coated withCHSE-214 cells.

[0221]FIG. 6 shows ISAV-specific antibodies in sera obtained fromAtlantic salmon infected with ISAV or rainbow trout injected with anucleic acid encoding an ISAV-specific DNA vaccine. Sera were collectedat 4, 6, 8, 10 and 12 weeks post-injection with 1 μg DNA vaccine orpost-infection with at least 1×10³ TCID₅₀ live ISAV/fish. Levels ofISAV-specific antibodies were expressed as a percentage of the mAbvalues to normalize variations between ELISA plates. ISAV-specificantibodies were detected at various times post-treatment. However, thelevels of ISAV-specific antibodies were much higher in fish that hadbeen exposed to live virus relative to those injected with the nucleicacid.

[0222] SDS-PAGE and Western Blot Analysis:

[0223] Whole cell lysates of naive and ISAV-infected CHSE cells as wellas purified ISAV were screened for the presence of immunoreactiveantigens with mAb 10A3 and sera from Atlantic salmon infected with ISAV.SDS-polyacrylamide gel electrophoresis (PAGE) was carried out by themethod of Laemmli (1970). Proteins were solubilized withSDS-polyacrylamide gel electrophoresis (PAGE) sample buffer andseparated by SDS-PAGE on 5% stacking gel and 12% resolving gel.Immunoreactive protein bands were visualized by Western blot analysis.Briefly, proteins separated by SDS-PAGE were electrophoreticallytransferred to nitrocellulose (Bio-Rad Laboratories). The membranes wereblocked with 3% skim milk buffer and then incubated with either mAb 10A3or sera from Atlantic salmon infected with ISAV followed by anincubation with goat anti-mouse immunoglobulin G conjugated to alkalinephosphatase or mouse anti-salmon immunoglobulin (Rob Beecroft),respectively. In the latter case, a final incubation with goatanti-mouse immunoglobulin G conjugated to alkaline phosphatase wasrequired. The immunoreactive proteins were visualized followingdevelopment with 5-bromo-4-chloro-3-indolyl phosphate and nitrobluetetrazolium.

[0224] Immunoreactive polypeptides encoded by the RNA segments wereidentified by Western blot analysis performed on naive and ISAV-infectedCHSE-214 cells (see Table 6 below). Sera, collected from Atlantic salmoninjected with live ISA virus, reacted with the 72 and 42 kDa proteins ofISA virus (Table 6). Similar analyses were performed with ISAvirus-specific mouse polyclonal and monoclonal antibodies. TABLE 6 ISAVimmunoreactive proteins detected by Western blot analysis Immunoreactiveproteins (kDa) Sera CHSE CHSE/ISAV Purified ISAV mAb 10A3 — 42 42 Mousepolyclonal — 42, 36, 25, 15, 42, 25, 15 11, 9 Atlantic salmon — 72, 42Not done convalescent

[0225] Of the six immunoreactive proteins present in the cellularpreparation of ISA virus and recognized by the mouse polyclonal sera,three were present in the purified ISA virus sample (42, 25 and 15 kDa;Table 5). Only the 42 kDa protein was recognized by the monoclonalantibody (Table 5). For each serum tested, no reaction was observed withthe naive CHSE sample indicating that the immunoreactive proteins werederived from ISA virus.

[0226] Serum Neutralization Assay

[0227] Ten-fold dilutions of ISAV in PBS were incubated with PBS orserum from naïve or ISAV-infected Atlantic salmon for 1 hr at 15° C.Aliquots of 100 μl of the serum/virus mixture were transferred inquadruplicate to 96-well cell culture plates seeded with CHSE-214 cells,incubated at 15° C. and monitored for CPE. Table 7 summarizes theresults. TABLE 7 Summary of serum neutralization studies SampleNeutralization ISAV/PBS − ISAV/sera from naïve Atlantic salmon −ISAV/sera from ISAV-infected Atlantic salmon +

[0228] Having illustrated and described the principals of the inventionby several embodiments, it should be apparent that those embodiments canbe modified in arrangement and detail without departing from theprinciples of the invention. Thus, the invention includes all suchembodiments and variations thereof, and their equivalents.

1 25 1 1749 DNA Infectious salmon anemia virus 1 ggatccccgg cgtttacttcttaaacacga aagaaatagt gactgcagaa gggaaagttg 60 atgaaacaag aggacccttagaaaggacat cagctcctct tatgagagat atctccaggc 120 tgatacaaga aacaatagaagaagtggaaa caggaggaga cccctctttt tcagtaagaa 180 gtgaaggagg ttctaaaatagaaggaagaa tcgccttttc attgcactca gaggtgtcca 240 cattgaaaat gaggatagcacttgaacaga aactggccag atatgagtac atgggagaaa 300 accttctcac acttgtcaaaaacacttcta tagacagaat gcagcctgat tctgcaatga 360 tggggaaaat ggtgttagaaagtcttagaa cacacacggt atcctctgag cagttgaatg 420 ggagaatgat tactgtccaatcgcaaggcc tagaaacaat agcgatatcg agtccttttg 480 atgtggaata cgacgatggatacgtattca caaggatgaa aggagacttt gtggcaatcg 540 gaagagacta caagggagctatcttatgct tcagagaagg acaagggaca ttcttcagtg 600 ggagaggcaa ctggtctgggctcatggaga gatgtctagt tgaaatgaga ctatgtccat 660 gcttttacag ctgcacctggcaagactacc ctgacaaaaa gagtctctac gaaaaagcaa 720 cgttcgaagc caaacagatagtctttgcta tgggagaaag tgttggagta gatgtcagag 780 taaacacaga tggtgaaataggagacaagg gaatttcctt actaacaagg gaaagagagg 840 acaaatacat gtcaaaagtgtcttatgagt gcagagtggt cagtgggaaa ctggtgatgg 900 gtttggacaa aatgagcagagtcgcaaaag ggaacctaga agtagtgagg gaaaaaggag 960 atgacacaag tcagtcagattccttttatg aaggtgtact acaggtaggt agcatgattg 1020 ggaccacaat ggagagtttaaaacagcagt tacaaggacc tgtgggaatt tggagagcat 1080 caggtgtttc agctatggaaaggtgcatga agagagggca aagcaaaact gtagtggcaa 1140 gtgctagata cacattccaaaagatgatgg aaaagatggc atctggtagg gaagtgtcta 1200 aatacagttt gataattgtcatgaggtgct gcattggttt cacgtctgaa gctaataaga 1260 gagctttaac taacatctctggaactggat actacattag tgttgcacaa cctaccgtag 1320 taaaactagc aggagaatggttgatcacac ctgtgggtag gtctaagaca ggggaagttc 1380 agtatgtatc tgccaagctgaagaagggga tgaccacagg gaagctagaa ttgattaaga 1440 aagcagatag atctgacttggacaacttcc cagaaccgtc ggctgatgaa ctgttgagag 1500 aaggaacaat tgtgttaatgcaaatcggaa aagacaaatg gttatgtagg gtaagaacag 1560 gtgataggag agtgaggaccgacacagaca tacagagggc agaagcaaaa tctcaggttg 1620 aaaaagaaga tttgatggatgaatatggtg tttaaaataa gtggttgtaa aaattgaatg 1680 ttgtttcttt tgctttttgagcctttgacg atacttttaa taaataaaat gtccattttg 1740 tccgatccc 1749 2 550PRT Infectious salmon anemia virus 2 Ile Pro Gly Val Tyr Phe Leu Asn ThrLys Glu Ile Val Thr Ala Glu 1 5 10 15 Gly Lys Val Asp Glu Thr Arg GlyPro Leu Glu Arg Thr Ser Ala Pro 20 25 30 Leu Met Arg Asp Ile Ser Arg LeuIle Gln Glu Thr Ile Glu Glu Val 35 40 45 Glu Thr Gly Gly Asp Pro Ser PheSer Val Arg Ser Glu Gly Gly Ser 50 55 60 Lys Ile Glu Gly Arg Ile Ala PheSer Leu His Ser Glu Val Ser Thr 65 70 75 80 Leu Lys Met Arg Ile Ala LeuGlu Gln Lys Leu Ala Arg Tyr Glu Tyr 85 90 95 Met Gly Glu Asn Leu Leu ThrLeu Val Lys Asn Thr Ser Ile Asp Arg 100 105 110 Met Gln Pro Asp Ser AlaMet Met Gly Lys Met Val Leu Glu Ser Leu 115 120 125 Arg Thr His Thr ValSer Ser Glu Gln Leu Asn Gly Arg Met Ile Thr 130 135 140 Val Gln Ser GlnGly Leu Glu Thr Ile Ala Ile Ser Ser Pro Phe Asp 145 150 155 160 Val GluTyr Asp Asp Gly Tyr Val Phe Thr Arg Met Lys Gly Asp Phe 165 170 175 ValAla Ile Gly Arg Asp Tyr Lys Gly Ala Ile Leu Cys Phe Arg Glu 180 185 190Gly Gln Gly Thr Phe Phe Ser Gly Arg Gly Asn Trp Ser Gly Leu Met 195 200205 Glu Arg Cys Leu Val Glu Met Arg Leu Cys Pro Cys Phe Tyr Ser Cys 210215 220 Thr Trp Gln Asp Tyr Pro Asp Lys Lys Ser Leu Tyr Glu Lys Ala Thr225 230 235 240 Phe Glu Ala Lys Gln Ile Val Phe Ala Met Gly Glu Ser ValGly Val 245 250 255 Asp Val Arg Val Asn Thr Asp Gly Glu Ile Gly Asp LysGly Ile Ser 260 265 270 Leu Leu Thr Arg Glu Arg Glu Asp Lys Tyr Met SerLys Val Ser Tyr 275 280 285 Glu Cys Arg Val Val Ser Gly Lys Leu Val MetGly Leu Asp Lys Met 290 295 300 Ser Arg Val Ala Lys Gly Asn Leu Glu ValVal Arg Glu Lys Gly Asp 305 310 315 320 Asp Thr Ser Gln Ser Asp Ser PheTyr Glu Gly Val Leu Gln Val Gly 325 330 335 Ser Met Ile Gly Thr Thr MetGlu Ser Leu Lys Gln Gln Leu Gln Gly 340 345 350 Pro Val Gly Ile Trp ArgAla Ser Gly Val Ser Ala Met Glu Arg Cys 355 360 365 Met Lys Arg Gly GlnSer Lys Thr Val Val Ala Ser Ala Arg Tyr Thr 370 375 380 Phe Gln Lys MetMet Glu Lys Met Ala Ser Gly Arg Glu Val Ser Lys 385 390 395 400 Tyr SerLeu Ile Ile Val Met Arg Cys Cys Ile Gly Phe Thr Ser Glu 405 410 415 AlaAsn Lys Arg Ala Leu Thr Asn Ile Ser Gly Thr Gly Tyr Tyr Ile 420 425 430Ser Val Ala Gln Pro Thr Val Val Lys Leu Ala Gly Glu Trp Leu Ile 435 440445 Thr Pro Val Gly Arg Ser Lys Thr Gly Glu Val Gln Tyr Val Ser Ala 450455 460 Lys Leu Lys Lys Gly Met Thr Thr Gly Lys Leu Glu Leu Ile Lys Lys465 470 475 480 Ala Asp Arg Ser Asp Leu Asp Asn Phe Pro Glu Pro Ser AlaAsp Glu 485 490 495 Leu Leu Arg Glu Gly Thr Ile Val Leu Met Gln Ile GlyLys Asp Lys 500 505 510 Trp Leu Cys Arg Val Arg Thr Gly Asp Arg Arg ValArg Thr Asp Thr 515 520 525 Asp Ile Gln Arg Ala Glu Ala Lys Ser Gln ValGlu Lys Glu Asp Leu 530 535 540 Met Asp Glu Tyr Gly Val 545 550 3 2185DNA Infectious salmon anemia virus 3 ccatggccgc gggattgaac gctctttaataaccatggaa actctagtag gagggctgct 60 gactggagaa gattctctga tcagtatgtcaaacgatgta tcttgtcttt atgtttacga 120 tggaccaatg agagttttct ctcagaacgcattaatgcca actctgcaaa gtgtaaaaag 180 aagtgaccaa ttttccaaag ggaaaacaaagagatttatc attgacctgt tcggaatgaa 240 gagaatgtgg gacatcggaa acaaacagttggaagacgag aacttagacg aaactgtagg 300 cgtggctgac ttggggctgg tgaaatatctaatcaacaac aagtacgatg aagcagaaaa 360 gacaagttta aggaagtcaa tggaagaagcattcgaaaaa tccatgaacg aagaatttgt 420 ggttttaaac aaaggaaagt ctgcaaacgacatcatttca gacacaaatg cgatgtgcaa 480 attctgtgta aagaactgga tagtggcaacaggtttcagg ggaagaacga tgtcagattt 540 aattgaacac catttcagat gcatgcaagggaaacaggag gtgaaaggat acatttggaa 600 acacaagtac aacgaaaggc ttaaaagaaaacagctaagc aaagaagaag tgaaattcga 660 cagagaagaa tatacttcaa gaagcttcagactactctct ttcttgaaga acagcgagag 720 gaccaaactc gagccgagag cagtgttcacagcaggagtt ccatggaggg cattcatctt 780 cgtcctagaa cagacaatgc tggtggtaaacaaactggac ccgaattcag tgatatggat 840 gggaagtgat gcaaagataa acaccacaaactccaggata aaggaaatag ggatgaaaaa 900 tcaaggacaa acactagtga cactcacaggagataactcc aaatacaacg agagcatgtg 960 cccagaggtg atgatggtgt tcctaagagaactaggaata aaaggaccaa tgttggaagt 1020 actggactat gcgctgtggc aattttcacagaagagtgta aaacctgtcg cacctataaa 1080 gaagagaacc ggcaagtcta ccgtggtgataaaagcagat tccgttaagg agtgtagaga 1140 tgccttcaac gaaaaggaac tggagctgattcaaggagtt gaatggatgg acgacggatt 1200 tgtgagagtg aggagaggaa tgttgatgggaatggcaaac aacgctttta ccacagcttc 1260 tacaattgcc tcctctttta gtttcacaccagaagctgtg tacacattac agagctcaga 1320 cgacttcgtt acaggtagct gtggaagagacgtgcaacac gcaagacaaa ggctagagat 1380 ggctcttaaa gtgagcaaag ccgcaggtctgaacgtatca cagaagaagt cattctacgt 1440 tgaagggaca actttcgagt tcaactctatgttcgtaaga gacggtaaag tgatggcaaa 1500 cggaggaaac tttgagaaca tgacagttcctggaggatta ggaccatcta cagatctctt 1560 tgtcgtgggg aaacaagcaa gaaactccatgttgagaggc aacctatcct tcagccaggc 1620 gatggagatg tgcaaaatag gaatcacaaatgttgagaaa gtttactatg gaaacagaaa 1680 ataccaggag ctgaaaaatg agataagagagaaatgtgga gaagaaacga tgtccatacc 1740 agagagcatg ggaggagaca ggaaaccaagaccgtgggaa ttacctcaga gctttgatgg 1800 aattgcctta aaagaagctg tgaacagaggacattggaaa gctgccaagt acatcaaatc 1860 ttgctgcagc atagagttcg atgaagaaggagaccaatct tgggacactt cgaaaacagc 1920 acttgtggtc ataaggaaaa atgaaacggacatgagaaga agaactgtta aaacgaggaa 1980 cccaaaagat aaaatcttca atgatgcaatgaacaaggcc aaaaggatgt acgaaacagt 2040 cgtggacaga aacccattac taggtctgaaggggaaggga ggtagactga cagtaaaaga 2100 cttgaaagca aggaagctta ttgatgaagtagaagttgtt aagaagaaaa agcatgtttg 2160 aaatcactag tgcggccgcc tgcag 2185 4726 PRT Infectious salmon anemia virus 4 His Gly Arg Gly Ile Glu Arg SerLeu Ile Thr Met Glu Thr Leu Val 1 5 10 15 Gly Gly Leu Leu Thr Gly GluAsp Ser Leu Ile Ser Met Ser Asn Asp 20 25 30 Val Ser Cys Leu Tyr Val TyrAsp Gly Pro Met Arg Val Phe Ser Gln 35 40 45 Asn Ala Leu Met Pro Thr LeuGln Ser Val Lys Arg Ser Asp Gln Phe 50 55 60 Ser Lys Gly Lys Thr Lys ArgPhe Ile Ile Asp Leu Phe Gly Met Lys 65 70 75 80 Arg Met Trp Asp Ile GlyAsn Lys Gln Leu Glu Asp Glu Asn Leu Asp 85 90 95 Glu Thr Val Gly Val AlaAsp Leu Gly Leu Val Lys Tyr Leu Ile Asn 100 105 110 Asn Lys Tyr Asp GluAla Glu Lys Thr Ser Leu Arg Lys Ser Met Glu 115 120 125 Glu Ala Phe GluLys Ser Met Asn Glu Glu Phe Val Val Leu Asn Lys 130 135 140 Gly Lys SerAla Asn Asp Ile Ile Ser Asp Thr Asn Ala Met Cys Lys 145 150 155 160 PheCys Val Lys Asn Trp Ile Val Ala Thr Gly Phe Arg Gly Arg Thr 165 170 175Met Ser Asp Leu Ile Glu His His Phe Arg Cys Met Gln Gly Lys Gln 180 185190 Glu Val Lys Gly Tyr Ile Trp Lys His Lys Tyr Asn Glu Arg Leu Lys 195200 205 Arg Lys Gln Leu Ser Lys Glu Glu Val Lys Phe Asp Arg Glu Glu Tyr210 215 220 Thr Ser Arg Ser Phe Arg Leu Leu Ser Phe Leu Lys Asn Ser GluArg 225 230 235 240 Thr Lys Leu Glu Pro Arg Ala Val Phe Thr Ala Gly ValPro Trp Arg 245 250 255 Ala Phe Ile Phe Val Leu Glu Gln Thr Met Leu ValVal Asn Lys Leu 260 265 270 Asp Pro Asn Ser Val Ile Trp Met Gly Ser AspAla Lys Ile Asn Thr 275 280 285 Thr Asn Ser Arg Ile Lys Glu Ile Gly MetLys Asn Gln Gly Gln Thr 290 295 300 Leu Val Thr Leu Thr Gly Asp Asn SerLys Tyr Asn Glu Ser Met Cys 305 310 315 320 Pro Glu Val Met Met Val PheLeu Arg Glu Leu Gly Ile Lys Gly Pro 325 330 335 Met Leu Glu Val Leu AspTyr Ala Leu Trp Gln Phe Ser Gln Lys Ser 340 345 350 Val Lys Pro Val AlaPro Ile Lys Lys Arg Thr Gly Lys Ser Thr Val 355 360 365 Val Ile Lys AlaAsp Ser Val Lys Glu Cys Arg Asp Ala Phe Asn Glu 370 375 380 Lys Glu LeuGlu Leu Ile Gln Gly Val Glu Trp Met Asp Asp Gly Phe 385 390 395 400 ValArg Val Arg Arg Gly Met Leu Met Gly Met Ala Asn Asn Ala Phe 405 410 415Thr Thr Ala Ser Thr Ile Ala Ser Ser Phe Ser Phe Thr Pro Glu Ala 420 425430 Val Tyr Thr Leu Gln Ser Ser Asp Asp Phe Val Thr Gly Ser Cys Gly 435440 445 Arg Asp Val Gln His Ala Arg Gln Arg Leu Glu Met Ala Leu Lys Val450 455 460 Ser Lys Ala Ala Gly Leu Asn Val Ser Gln Lys Lys Ser Phe TyrVal 465 470 475 480 Glu Gly Thr Thr Phe Glu Phe Asn Ser Met Phe Val ArgAsp Gly Lys 485 490 495 Val Met Ala Asn Gly Gly Asn Phe Glu Asn Met ThrVal Pro Gly Gly 500 505 510 Leu Gly Pro Ser Thr Asp Leu Phe Val Val GlyLys Gln Ala Arg Asn 515 520 525 Ser Met Leu Arg Gly Asn Leu Ser Phe SerGln Ala Met Glu Met Cys 530 535 540 Lys Ile Gly Ile Thr Asn Val Glu LysVal Tyr Tyr Gly Asn Arg Lys 545 550 555 560 Tyr Gln Glu Leu Lys Asn GluIle Arg Glu Lys Cys Gly Glu Glu Thr 565 570 575 Met Ser Ile Pro Glu SerMet Gly Gly Asp Arg Lys Pro Arg Pro Trp 580 585 590 Glu Leu Pro Gln SerPhe Asp Gly Ile Ala Leu Lys Glu Ala Val Asn 595 600 605 Arg Gly His TrpLys Ala Ala Lys Tyr Ile Lys Ser Cys Cys Ser Ile 610 615 620 Glu Phe AspGlu Glu Gly Asp Gln Ser Trp Asp Thr Ser Lys Thr Ala 625 630 635 640 LeuVal Val Ile Arg Lys Asn Glu Thr Asp Met Arg Arg Arg Thr Val 645 650 655Lys Thr Arg Asn Pro Lys Asp Lys Ile Phe Asn Asp Ala Met Asn Lys 660 665670 Ala Lys Arg Met Tyr Glu Thr Val Val Asp Arg Asn Pro Leu Leu Gly 675680 685 Leu Lys Gly Lys Gly Gly Arg Leu Thr Val Lys Asp Leu Lys Ala Arg690 695 700 Lys Leu Ile Asp Glu Val Glu Val Val Lys Lys Lys Lys His ValAsn 705 710 715 720 His Cys Gly Arg Leu Gln 725 5 2046 DNA Infectioussalmon anemia virus 5 agcaaagatt gctcaaatcc caaaaataat acagaaaacgtataagagat ggccgataaa 60 ggtatgactt attcttttga tgtcagagac aacaccttggttgtaagaag atctaccgct 120 actaaaagtg gcattaagat ctcctacaga gaggatcgaggaacatcact tctccaaaag 180 gcattcgccg ggacagaaga tgaattctgg gtggagttagatcaagatgt ctacgttgac 240 aaaaagatta gagaattcct ggtagaagag aaaatgaaggacatgagcac aagagtgtct 300 ggggcagtgg cagcagcaat tgaaagatca gttgaatttgacaatttctc aaaagaagca 360 gcagctaaca ttgaaatggc tggtgtagat gatgaagaagctggaggaag tggtctggta 420 gacaacagaa ggaagaacaa aggggtctca aacatggcctacaatctgtc tctattcata 480 gggatggtgt ttcctgctct cactactttc ttcagtgctatcctatcaga aggtgaaatg 540 agcatctggc aaaatggaca agcaatcatg agaattctggcactggcaga tgaagacgga 600 aagagacaaa caagaacagg aggacagagg gtggacatggctgatgtaac caagctgaac 660 gtagtcacgg ctaacggaaa agtcaagcaa gttgaagtaaacttgaacga tctcaaagca 720 gcattcaggc agagtagacc taaaagatcg gactacagaaaagggcaagg ttccaaggct 780 acagaatcaa gcatctccaa ccaatgtatg gcactgattatgaaatctgt gctgtcagca 840 gaccaacttt ttgctccggg agtgaagatg atgaggacgaacggtttcaa tgcgtcgtac 900 acaacactgg cagaaggggc aaacattccg agcaagtacctaagacacat gaggaactgc 960 ggaggagtag ctctggacct gatgggaatg aagaggatcaaaaactcacc tgaaggagcc 1020 aagtctaaga tcttttccat catccagaag aaagtaagaggaagatgtcg cacagaggag 1080 caacgcctcc tgactagcgc actgaaaatc agcgacggtgaaaacaagtt ccagagaatc 1140 atggacactc tatgtacaag cttcctgatt gaccctccaagaactaccaa atgcttcatt 1200 ccacctattt ccagtctcat gatgtacatc caagaaggcaactctgtact ggcaatggat 1260 ttcatgaaaa acggagagga cgcctgcaag atctgcagagaagccaaact gaaagtgggg 1320 gtaaacagta cgttcacaat gtcagtagct agaacatgcgttgcagtgtc aatggttgca 1380 acagcttttt gttctgcaga tatcatcgag aatgcagtgcctggttccga aaggtacaga 1440 tccaacatca aggctaacac aaccaaacca aaaaaggactccacttacac aattcaagga 1500 cttagattgt ctaacgtgag gtatgaagca agacctgaaacatcacaaag caacacagac 1560 agaagttggc aagtgaacgt gactgacagc ttcggaggacttgctgtgtt caaccaaggg 1620 gcaattagag aaatgctagg agacggaaca tcagagacaactagtgtgaa cgtcagagcc 1680 ctggtgaaga gaattctgaa atcagcttca gagaggagtgcaagagctgt aaagacattt 1740 atggtgggag aacaagggaa atcagctatt gttatctctggtgtgggact gttctctatt 1800 gactttgaag gggtagagga agcggaaaga ataactgacatgacacctga aattgagttt 1860 gacgaggacg acgaggaaga ggaagacatt gacatttagagtgacaatta tgtaacttcc 1920 taattaccct atattgtttg aatatataat gaaactattgtgtgttaaag gttgtgggtt 1980 tgattattaa atttaaattg aaacggtatt gacgatatttacaaaaaaaa aaaaaaaaaa 2040 aaaaaa 2046 6 616 PRT Infectious salmonanemia virus 6 Met Ala Asp Lys Gly Met Thr Tyr Ser Phe Asp Val Arg AspAsn Thr 1 5 10 15 Leu Val Val Arg Arg Ser Thr Ala Thr Lys Ser Gly IleLys Ile Ser 20 25 30 Tyr Arg Glu Asp Arg Gly Thr Ser Leu Leu Gln Lys AlaPhe Ala Gly 35 40 45 Thr Glu Asp Glu Phe Trp Val Glu Leu Asp Gln Asp ValTyr Val Asp 50 55 60 Lys Lys Ile Arg Glu Phe Leu Val Glu Glu Lys Met LysAsp Met Ser 65 70 75 80 Thr Arg Val Ser Gly Ala Val Ala Ala Ala Ile GluArg Ser Val Glu 85 90 95 Phe Asp Asn Phe Ser Lys Glu Ala Ala Ala Asn IleGlu Met Ala Gly 100 105 110 Val Asp Asp Glu Glu Ala Gly Gly Ser Gly LeuVal Asp Asn Arg Arg 115 120 125 Lys Asn Lys Gly Val Ser Asn Met Ala TyrAsn Leu Ser Leu Phe Ile 130 135 140 Gly Met Val Phe Pro Ala Leu Thr ThrPhe Phe Ser Ala Ile Leu Ser 145 150 155 160 Glu Gly Glu Met Ser Ile TrpGln Asn Gly Gln Ala Ile Met Arg Ile 165 170 175 Leu Ala Leu Ala Asp GluAsp Gly Lys Arg Gln Thr Arg Thr Gly Gly 180 185 190 Gln Arg Val Asp MetAla Asp Val Thr Lys Leu Asn Val Val Thr Ala 195 200 205 Asn Gly Lys ValLys Gln Val Glu Val Asn Leu Asn Asp Leu Lys Ala 210 215 220 Ala Phe ArgGln Ser Arg Pro Lys Arg Ser Asp Tyr Arg Lys Gly Gln 225 230 235 240 GlySer Lys Ala Thr Glu Ser Ser Ile Ser Asn Gln Cys Met Ala Leu 245 250 255Ile Met Lys Ser Val Leu Ser Ala Asp Gln Leu Phe Ala Pro Gly Val 260 265270 Lys Met Met Arg Thr Asn Gly Phe Asn Ala Ser Tyr Thr Thr Leu Ala 275280 285 Glu Gly Ala Asn Ile Pro Ser Lys Tyr Leu Arg His Met Arg Asn Cys290 295 300 Gly Gly Val Ala Leu Asp Leu Met Gly Met Lys Arg Ile Lys AsnSer 305 310 315 320 Pro Glu Gly Ala Lys Ser Lys Ile Phe Ser Ile Ile GlnLys Lys Val 325 330 335 Arg Gly Arg Cys Arg Thr Glu Glu Gln Arg Leu LeuThr Ser Ala Leu 340 345 350 Lys Ile Ser Asp Gly Glu Asn Lys Phe Gln ArgIle Met Asp Thr Leu 355 360 365 Cys Thr Ser Phe Leu Ile Asp Pro Pro ArgThr Thr Lys Cys Phe Ile 370 375 380 Pro Pro Ile Ser Ser Leu Met Met TyrIle Gln Glu Gly Asn Ser Val 385 390 395 400 Leu Ala Met Asp Phe Met LysAsn Gly Glu Asp Ala Cys Lys Ile Cys 405 410 415 Arg Glu Ala Lys Leu LysVal Gly Val Asn Ser Thr Phe Thr Met Ser 420 425 430 Val Ala Arg Thr CysVal Ala Val Ser Met Val Ala Thr Ala Phe Cys 435 440 445 Ser Ala Asp IleIle Glu Asn Ala Val Pro Gly Ser Glu Arg Tyr Arg 450 455 460 Ser Asn IleLys Ala Asn Thr Thr Lys Pro Lys Lys Asp Ser Thr Tyr 465 470 475 480 ThrIle Gln Gly Leu Arg Leu Ser Asn Val Arg Tyr Glu Ala Arg Pro 485 490 495Glu Thr Ser Gln Ser Asn Thr Asp Arg Ser Trp Gln Val Asn Val Thr 500 505510 Asp Ser Phe Gly Gly Leu Ala Val Phe Asn Gln Gly Ala Ile Arg Glu 515520 525 Met Leu Gly Asp Gly Thr Ser Glu Thr Thr Ser Val Asn Val Arg Ala530 535 540 Leu Val Lys Arg Ile Leu Lys Ser Ala Ser Glu Arg Ser Ala ArgAla 545 550 555 560 Val Lys Thr Phe Met Val Gly Glu Gln Gly Lys Ser AlaIle Val Ile 565 570 575 Ser Gly Val Gly Leu Phe Ser Ile Asp Phe Glu GlyVal Glu Glu Ala 580 585 590 Glu Arg Ile Thr Asp Met Thr Pro Glu Ile GluPhe Asp Glu Asp Asp 595 600 605 Glu Glu Glu Glu Asp Ile Asp Ile 610 6157 1787 DNA Infectious salmon anemia virus 7 caagatggat aacctccgtgaatgcataaa ccgcaaaaga agactacttg ccttaccaga 60 tgttcctgaa acttcggatgcctttctaag tgatttgaga catctataca tgtgtgttgc 120 tttctgtgat caacacaaaaccactggaga cgaatcaaga ttcaccaacc tggaattact 180 tgaccaagat gaagcactaggtgcccaaag agcttttgaa gccaaacatg gaataaaagg 240 aggttcttta ggagacgttcttgaccatga actgaaaaag gtcattgaat ttacttttac 300 ttctggaagt ttgtatattgccgaacaaag aaaaagaaag actcaagcag actcaataat 360 tgtgtgcgtt tcagaaggacttaacgactt cagcgtatca cacggagtgc tagacatggg 420 acttgtggaa acaggggtgaatgcagtaag agatttctgc acacaaaacg gaataccaat 480 gaagataaat caggtaggatccacgagaac accaacaccg atcagcacat gcaaaatctc 540 tgaacaaata acacgacarataaacagtac aattactgaa aggaaaatgg aaacagtact 600 ggcagcaatc gcaattaaaccagaactcaa ayyaactcag aaaggatgca gmmcttgtaa 660 agaactagaa gatgaaaatattctgtggat ggaccctcaa ttctgtgaaa ttgatgaaag 720 ttttccttac agaggagggccatacgggaa cttcctgcaa gaattgctgc ttacaaccaa 780 cgacgtagag accaacgggaaagacagaga agaagtagta aagaasatac tggataacaa 840 ggcgttcacc gttgaaagtggtgaatgcat aataacactt ccagacaaaa tgacttgttt 900 cggagaacar gagaagaagagaccagcaac aatagacgaa gtgagaaccg caggagaaag 960 gtttgaacag agtgttaaaccgaaaaccca aagatatgga aggttatcag acaaatggat 1020 ggagcttgaa aagtttatctttactgcaag caaaacagaa gtggatactt tcctttctgt 1080 agggaccgaa agacttgagtcggttggagt gtgtgtcgga gctttacaca gagcgaccac 1140 aaccaggata attagacctatgattcaagg agggaaatgt tgggggatga tgttcaaaac 1200 aaagtccaaa atgggagacacgaggaagga aggatactgt cacgcaatca ttttcggaaa 1260 aggggaagat aaatcaggacaaaacaagat gacaatgatg gggaaaacag tacattggca 1320 tctaagagta gttaagtctaaaggagactg gatggcgcaa caactctgtg caaacaaaag 1380 cagaatatgg caacatgaccctgagctagt aacagaagga gtgacagttc taatgacgcc 1440 tttttctcag aaaattgcaaccattagtag atggagggca atgaggttag acagcatgtt 1500 tcatgtttct agtgcctggcatcattcacc tgcgtgtgaa gctgcatcgg caatgctgag 1560 aaagtttgtg gagatagtacatgccatcaa ccagaaaaga gattggggtg ttgtggggag 1620 tatggaggac atggtgaaggaagtggagga aataggggag cacttgcaga cggcatgtga 1680 ytttagagtt tacaacatktgcaaagcctt gattcagaaa attgcagtca gtacccaatg 1740 agtggttatt tacttgtaaattgttgtgtg tttgacgata tgtattt 1787 8 578 PRT Infectious salmon anemiavirus misc_feature (210)..(210) Xaa can be any naturally occurring aminoacid 8 Met Asp Asn Leu Arg Glu Cys Ile Asn Arg Lys Arg Arg Leu Leu Ala 15 10 15 Leu Pro Asp Val Pro Glu Thr Ser Asp Ala Phe Leu Ser Asp Leu Arg20 25 30 His Leu Tyr Met Cys Val Ala Phe Cys Asp Gln His Lys Thr Thr Gly35 40 45 Asp Glu Ser Arg Phe Thr Asn Leu Glu Leu Leu Asp Gln Asp Glu Ala50 55 60 Leu Gly Ala Gln Arg Ala Phe Glu Ala Lys His Gly Ile Lys Gly Gly65 70 75 80 Ser Leu Gly Asp Val Leu Asp His Glu Leu Lys Lys Val Ile GluPhe 85 90 95 Thr Phe Thr Ser Gly Ser Leu Tyr Ile Ala Glu Gln Arg Lys ArgLys 100 105 110 Thr Gln Ala Asp Ser Ile Ile Val Cys Val Ser Glu Gly LeuAsn Asp 115 120 125 Phe Ser Val Ser His Gly Val Leu Asp Met Gly Leu ValGlu Thr Gly 130 135 140 Val Asn Ala Val Arg Asp Phe Cys Thr Gln Asn GlyIle Pro Met Lys 145 150 155 160 Ile Asn Gln Val Gly Ser Thr Arg Thr ProThr Pro Ile Ser Thr Cys 165 170 175 Lys Ile Ser Glu Gln Ile Thr Arg GlnIle Asn Ser Thr Ile Thr Glu 180 185 190 Arg Lys Met Glu Thr Val Leu AlaAla Ile Ala Ile Lys Pro Glu Leu 195 200 205 Lys Xaa Thr Gln Lys Gly CysXaa Xaa Cys Lys Glu Leu Glu Asp Glu 210 215 220 Asn Ile Leu Trp Met AspPro Gln Phe Cys Glu Ile Asp Glu Ser Phe 225 230 235 240 Pro Tyr Arg GlyGly Pro Tyr Gly Asn Phe Leu Gln Glu Leu Leu Leu 245 250 255 Thr Thr AsnAsp Val Glu Thr Asn Gly Lys Asp Arg Glu Glu Val Val 260 265 270 Lys XaaIle Leu Asp Asn Lys Ala Phe Thr Val Glu Ser Gly Glu Cys 275 280 285 IleIle Thr Leu Pro Asp Lys Met Thr Cys Phe Gly Glu Gln Glu Lys 290 295 300Lys Arg Pro Ala Thr Ile Asp Glu Val Arg Thr Ala Gly Glu Arg Phe 305 310315 320 Glu Gln Ser Val Lys Pro Lys Thr Gln Arg Tyr Gly Arg Leu Ser Asp325 330 335 Lys Trp Met Glu Leu Glu Lys Phe Ile Phe Thr Ala Ser Lys ThrGlu 340 345 350 Val Asp Thr Phe Leu Ser Val Gly Thr Glu Arg Leu Glu SerVal Gly 355 360 365 Val Cys Val Gly Ala Leu His Arg Ala Thr Thr Thr ArgIle Ile Arg 370 375 380 Pro Met Ile Gln Gly Gly Lys Cys Trp Gly Met MetPhe Lys Thr Lys 385 390 395 400 Ser Lys Met Gly Asp Thr Arg Lys Glu GlyTyr Cys His Ala Ile Ile 405 410 415 Phe Gly Lys Gly Glu Asp Lys Ser GlyGln Asn Lys Met Thr Met Met 420 425 430 Gly Lys Thr Val His Trp His LeuArg Val Val Lys Ser Lys Gly Asp 435 440 445 Trp Met Ala Gln Gln Leu CysAla Asn Lys Ser Arg Ile Trp Gln His 450 455 460 Asp Pro Glu Leu Val ThrGlu Gly Val Thr Val Leu Met Thr Pro Phe 465 470 475 480 Ser Gln Lys IleAla Thr Ile Ser Arg Trp Arg Ala Met Arg Leu Asp 485 490 495 Ser Met PheHis Val Ser Ser Ala Trp His His Ser Pro Ala Cys Glu 500 505 510 Ala AlaSer Ala Met Leu Arg Lys Phe Val Glu Ile Val His Ala Ile 515 520 525 AsnGln Lys Arg Asp Trp Gly Val Val Gly Ser Met Glu Asp Met Val 530 535 540Lys Glu Val Glu Glu Ile Gly Glu His Leu Gln Thr Ala Cys Asp Phe 545 550555 560 Arg Val Tyr Asn Xaa Cys Lys Ala Leu Ile Gln Lys Ile Ala Val Ser565 570 575 Thr Gln 9 1504 DNA Infectious salmon anemia virus 9agttaaagat ggcttttcta acaattttag tcttgttcct ttttaaagag gttctttgtg 60aaccttgtat ttgtgagaac ccaacatgtc taggaataac aatcccacag gcaggtttcg 120taagaagcgc tccaggaggt gtacttctaa ctgagacaat cacggaaaga ccacaactaa 180cagagtggac aacctccaga ccgaagcttg aagaaactct ctggttagat ggggaaacaa 240agaacggaaa agtatctcag acactattcg aagccatcca aggtacacag atggagaact 300gtgcagtgaa agctgtgtta gacacaacat ttgtcaacct aaccaaacaa gacattgtgc 360taggaaaaat caaggtgtct gagtttggtg gagacagtga catttccaaa tgtggaagaa 420aaggactaaa ggttttcatc tgtggaggta ctgttggata cgtgacaaga ggatgcccac 480ctgaggagtg caaaggaaag aaagggagaa tgatggctct cgaacccact acggattgtg 540gtgtcgaaaa aggacttaca actgacagaa tcaaaacagg aatgttggac atcacaagtt 600gctgtacaca acatggatgc acaaagggaa tcagagtaga ggttccttca ccagtacttg 660tatcttcaaa atgtcaagaa gtcactttca gagtggttcc attccattca gtacctgaca 720agctagggtt tgcacgcaca agctcattca cactaaaagc taacttcgtg aacaaacatg 780ggtggtccaa gtataatttc aacctaagag gatttcctgg agaagagttc attaagtgtt 840gtggatttac gttgggagtc ggaggagcgt ggtttcaagc ctacttaaat ggaatggttc 900aaggtgacgg tgccgcatct gcagacgacg tgaaagagaa actcaacgga ataatcgacc 960agataaacaa agcgaacaca cttcttgaag gagaaattga agcagtgagg aggattgcct 1020atatgaacca agcatcaagt cttcagaacc aagtggaaat cggactaata ggtgaatatt 1080tgaacattag cagttggttg gagactacta cattaactaa aacagaagaa ggcttgatga 1140agaatggctg gtgtcagtct aacacgcact gctggtgtcc acctaaacct acaattgttc 1200ccaccattgg atatgttgac agtataaaag aagtaacggg tacaagttgg tggatggtta 1260tgatacatta cattattgtg gggttaatag ttattgtggt ggtggtgttt ggtttaaaac 1320tatggggatg tcttagaagg tgaaatgtcg gtctaaaaat tctttttctg tacattacta 1380aagggtagct taaccaaggt gtttatgtat atagactatt attggataag ttagaaattt 1440gtatctgatt atgcattatt aattgtataa atagaatcac tagtgcggcc gcctgcaggt 1500cgac 1504 10 497 PRT Infectious salmon anemia virus 10 Leu Lys Met AlaPhe Leu Thr Ile Leu Val Leu Phe Leu Phe Lys Glu 1 5 10 15 Val Leu CysGlu Pro Cys Ile Cys Glu Asn Pro Thr Cys Leu Gly Ile 20 25 30 Thr Ile ProGln Ala Gly Phe Val Arg Ser Ala Pro Gly Gly Val Leu 35 40 45 Leu Thr GluThr Ile Thr Glu Arg Pro Gln Leu Thr Glu Trp Thr Thr 50 55 60 Ser Arg ProLys Leu Glu Glu Thr Leu Trp Leu Asp Gly Glu Thr Lys 65 70 75 80 Asn GlyLys Val Ser Gln Thr Leu Phe Glu Ala Ile Gln Gly Thr Gln 85 90 95 Met GluAsn Cys Ala Val Lys Ala Val Leu Asp Thr Thr Phe Val Asn 100 105 110 LeuThr Lys Gln Asp Ile Val Leu Gly Lys Ile Lys Val Ser Glu Phe 115 120 125Gly Gly Asp Ser Asp Ile Ser Lys Cys Gly Arg Lys Gly Leu Lys Val 130 135140 Phe Ile Cys Gly Gly Thr Val Gly Tyr Val Thr Arg Gly Cys Pro Pro 145150 155 160 Glu Glu Cys Lys Gly Lys Lys Gly Arg Met Met Ala Leu Glu ProThr 165 170 175 Thr Asp Cys Gly Val Glu Lys Gly Leu Thr Thr Asp Arg IleLys Thr 180 185 190 Gly Met Leu Asp Ile Thr Ser Cys Cys Thr Gln His GlyCys Thr Lys 195 200 205 Gly Ile Arg Val Glu Val Pro Ser Pro Val Leu ValSer Ser Lys Cys 210 215 220 Gln Glu Val Thr Phe Arg Val Val Pro Phe HisSer Val Pro Asp Lys 225 230 235 240 Leu Gly Phe Ala Arg Thr Ser Ser PheThr Leu Lys Ala Asn Phe Val 245 250 255 Asn Lys His Gly Trp Ser Lys TyrAsn Phe Asn Leu Arg Gly Phe Pro 260 265 270 Gly Glu Glu Phe Ile Lys CysCys Gly Phe Thr Leu Gly Val Gly Gly 275 280 285 Ala Trp Phe Gln Ala TyrLeu Asn Gly Met Val Gln Gly Asp Gly Ala 290 295 300 Ala Ser Ala Asp AspVal Lys Glu Lys Leu Asn Gly Ile Ile Asp Gln 305 310 315 320 Ile Asn LysAla Asn Thr Leu Leu Glu Gly Glu Ile Glu Ala Val Arg 325 330 335 Arg IleAla Tyr Met Asn Gln Ala Ser Ser Leu Gln Asn Gln Val Glu 340 345 350 IleGly Leu Ile Gly Glu Tyr Leu Asn Ile Ser Ser Trp Leu Glu Thr 355 360 365Thr Thr Leu Thr Lys Thr Glu Glu Gly Leu Met Lys Asn Gly Trp Cys 370 375380 Gln Ser Asn Thr His Cys Trp Cys Pro Pro Lys Pro Thr Ile Val Pro 385390 395 400 Thr Ile Gly Tyr Val Asp Ser Ile Lys Glu Val Thr Gly Thr SerTrp 405 410 415 Trp Met Val Met Ile His Tyr Ile Ile Val Gly Leu Ile ValIle Val 420 425 430 Val Val Val Phe Gly Leu Lys Leu Trp Gly Cys Leu ArgArg Asn Val 435 440 445 Gly Leu Lys Ile Leu Phe Leu Tyr Ile Thr Lys GlyLeu Asn Gln Gly 450 455 460 Val Tyr Val Tyr Arg Leu Leu Leu Asp Lys LeuGlu Ile Cys Ile Leu 465 470 475 480 Cys Ile Ile Asn Cys Ile Asn Arg IleThr Ser Ala Ala Ala Cys Arg 485 490 495 Ser 11 1323 DNA Infectioussalmon anemia virus 11 agcaaagatg gcacgattca taattttatt cctactgttggcgcctgttt acagtcgtct 60 atgtcttaga aaccatcctg acaccacctg gataggtgactcccgaagcg atcaatcaag 120 ggtgaaccaa cagtctcttg atctggttac aaacttcaagggaattctac aagccaagaa 180 cgggaatggt ctcatgaagc agatgagcgg aaggttcccaagtgattggt accaacctac 240 tacaaagtat aggattctat acattggtac aaacgactgcactgagggcc ctaacgacgt 300 gatcataccg acgtcaatga cactagacaa tgtggcaagggacctgtacc tgggagcatg 360 tcgaggagat gtaagagtga caccaacctt cgtgggagcagctgagcttg gactgattgg 420 gagaacagat gccttaacag aattttctgt aaaggtgctgactttcaaca accctactat 480 tgtagtagtt ggactaaatg gaatgtcagg aatctacaaggtctgcattg ctgcctcttc 540 tggaaacgta ggcggagtca acttggtgaa cggatgcggatacttcagcg ctcctctgag 600 attcgacaac ttcaaaggac agatctacgt gtcagacacctttgaagtca gaggaacaaa 660 gaacaaatgt gtcatactta gatcttctag caatgctcctttgtgtacac atatcaaaag 720 aaacattgag ttggatgagt acgttgacac accaaacactgggggcgtat atccttctga 780 tgggtttgat tctcttcacg gctctgcttc gattagaacttttttaacag aggcactgac 840 atgtccaggt gtagattggg acagaattga tgcagcttcatgcgagtatg acagttgtcc 900 taaacttgtg aaagaatttg accaaacagg gctcggaaacacagatactc aaataatgag 960 agagctagaa gcacaaaagg agatgattgg taaacttggcagaaacatta cagacgtaaa 1020 caacagagta gatgctattc caccacagct tagcaacatcttcatctcta tgggagtggc 1080 aggttttggg atagcactgt ttctagcagg gtggaaggcttgtgtttgga tagcagcttt 1140 catgtataag tctagaggta gaaacccacc tgcaaatctgtctgttgctt gatactaaga 1200 caaacaaagt tttcaaataa tcaaatgttt tctaatgtaatgtaaaattc aaatcgtatg 1260 tgatattatt attttgaaga cgttcttgat gttgtacgtttacagaaaag tgcatttttt 1320 act 1323 12 394 PRT Infectious salmon anemiavirus 12 Met Ala Arg Phe Ile Ile Leu Phe Leu Leu Leu Ala Pro Val Tyr Ser1 5 10 15 Arg Leu Cys Leu Arg Asn His Pro Asp Thr Thr Trp Ile Gly AspSer 20 25 30 Arg Ser Asp Gln Ser Arg Val Asn Gln Gln Ser Leu Asp Leu ValThr 35 40 45 Asn Phe Lys Gly Ile Leu Gln Ala Lys Asn Gly Asn Gly Leu MetLys 50 55 60 Gln Met Ser Gly Arg Phe Pro Ser Asp Trp Tyr Gln Pro Thr ThrLys 65 70 75 80 Tyr Arg Ile Leu Tyr Ile Gly Thr Asn Asp Cys Thr Glu GlyPro Asn 85 90 95 Asp Val Ile Ile Pro Thr Ser Met Thr Leu Asp Asn Val AlaArg Asp 100 105 110 Leu Tyr Leu Gly Ala Cys Arg Gly Asp Val Arg Val ThrPro Thr Phe 115 120 125 Val Gly Ala Ala Glu Leu Gly Leu Ile Gly Arg ThrAsp Ala Leu Thr 130 135 140 Glu Phe Ser Val Lys Val Leu Thr Phe Asn AsnPro Thr Ile Val Val 145 150 155 160 Val Gly Leu Asn Gly Met Ser Gly IleTyr Lys Val Cys Ile Ala Ala 165 170 175 Ser Ser Gly Asn Val Gly Gly ValAsn Leu Val Asn Gly Cys Gly Tyr 180 185 190 Phe Ser Ala Pro Leu Arg PheAsp Asn Phe Lys Gly Gln Ile Tyr Val 195 200 205 Ser Asp Thr Phe Glu ValArg Gly Thr Lys Asn Lys Cys Val Ile Leu 210 215 220 Arg Ser Ser Ser AsnAla Pro Leu Cys Thr His Ile Lys Arg Asn Ile 225 230 235 240 Glu Leu AspGlu Tyr Val Asp Thr Pro Asn Thr Gly Gly Val Tyr Pro 245 250 255 Ser AspGly Phe Asp Ser Leu His Gly Ser Ala Ser Ile Arg Thr Phe 260 265 270 LeuThr Glu Ala Leu Thr Cys Pro Gly Val Asp Trp Asp Arg Ile Asp 275 280 285Ala Ala Ser Cys Glu Tyr Asp Ser Cys Pro Lys Leu Val Lys Glu Phe 290 295300 Asp Gln Thr Gly Leu Gly Asn Thr Asp Thr Gln Ile Met Arg Glu Leu 305310 315 320 Glu Ala Gln Lys Glu Met Ile Gly Lys Leu Gly Arg Asn Ile ThrAsp 325 330 335 Val Asn Asn Arg Val Asp Ala Ile Pro Pro Gln Leu Ser AsnIle Phe 340 345 350 Ile Ser Met Gly Val Ala Gly Phe Gly Ile Ala Leu PheLeu Ala Gly 355 360 365 Trp Lys Ala Cys Val Trp Ile Ala Ala Phe Met TyrLys Ser Arg Gly 370 375 380 Arg Asn Pro Pro Ala Asn Leu Ser Val Ala 385390 13 966 DNA Infectious salmon anemia virus 13 tacaaagaaa atgttcagaacatgtctgga tttaacttcg aggtaatggt gccggaacaa 60 ggaggaaaag tggtcttcagccttactgaa acggggtcat gtgtctcgtt ttacggagat 120 gatgaaccag gtgaagggtcctgcgaactt gcctctgaaa acatggattt tccaagttgt 180 cctctgggga atggagatgacttctgtctg tcgctggcgc taagcacaat gagatggtct 240 gggatgacca agagaaacaacttcatggac agattcattg gaagttttgt tcattgtaca 300 ccagtgatga tctggtcgtatggaaatttg tccaagaaaa gccatcacaa aatggtttgc 360 cacacttgcc cagacgagtacaagttcagt gacaaggacg agatgcaggg atactatgag 420 gaatgtctag aggcttctactgacattttc cttgatgaac ttgctactgt tgttacaggt 480 ggcttctttc ctgtcggactcaaaggttcc tggggaggat ggtacctcaa gtacgtcagg 540 tatgctggac ctcttgcgggatcaagtgga ttcattgtca atcaacgatt ctacgacaga 600 gcccaaaaca agactggatccagggttgta tccatggttg aaatggacgg agacggctta 660 tcgttcatct acgagaagcctagcgtctac catagtgatg ggtgcactgg ttcagcagcg 720 aggttctgga aacgggatcacaatgagaga gctggagttg agcttagggc tggacttcac 780 ttcagaatgt gattggttgaaaacttgtta tgtaaacaag aattttgtgt ttttgtcaga 840 aaaagaaatt gctgtaaacatggaagttga aaaattcatt tgtaatgaga actaaagatg 900 tctttgtgtt caaattttaactaatgacaa tatatgaaat atgtcgtaca tggtgttgat 960 gataat 966 14 256 PRTInfectious salmon anemia virus 14 Met Ser Gly Phe Asn Phe Glu Val MetVal Pro Glu Gln Gly Gly Lys 1 5 10 15 Val Val Phe Ser Leu Thr Glu ThrGly Ser Cys Val Ser Phe Tyr Gly 20 25 30 Asp Asp Glu Pro Gly Glu Gly SerCys Glu Leu Ala Ser Glu Asn Met 35 40 45 Asp Phe Pro Ser Cys Pro Leu GlyAsn Gly Asp Asp Phe Cys Leu Ser 50 55 60 Leu Ala Leu Ser Thr Met Arg TrpSer Gly Met Thr Lys Arg Asn Asn 65 70 75 80 Phe Met Asp Arg Phe Ile GlySer Phe Val His Cys Thr Pro Val Met 85 90 95 Ile Trp Ser Tyr Gly Asn LeuSer Lys Lys Ser His His Lys Met Val 100 105 110 Cys His Thr Cys Pro AspGlu Tyr Lys Phe Ser Asp Lys Asp Glu Met 115 120 125 Gln Gly Tyr Tyr GluGlu Cys Leu Glu Ala Ser Thr Asp Ile Phe Leu 130 135 140 Asp Glu Leu AlaThr Val Val Thr Gly Gly Phe Phe Pro Val Gly Leu 145 150 155 160 Lys GlySer Trp Gly Gly Trp Tyr Leu Lys Tyr Val Arg Tyr Ala Gly 165 170 175 ProLeu Ala Gly Ser Ser Gly Phe Ile Val Asn Gln Arg Phe Tyr Asp 180 185 190Arg Ala Gln Asn Lys Thr Gly Ser Arg Val Val Ser Met Val Glu Met 195 200205 Asp Gly Asp Gly Leu Ser Phe Ile Tyr Glu Lys Pro Ser Val Tyr His 210215 220 Ser Asp Gly Cys Thr Gly Ser Ala Ala Arg Phe Trp Lys Arg Asp His225 230 235 240 Asn Glu Arg Ala Gly Val Glu Leu Arg Ala Gly Leu His PheArg Met 245 250 255 15 146 PRT Infectious salmon anemia virus 15 Met AsnLeu Leu Leu Leu Leu Gln Val Ala Ser Phe Leu Ser Asp Ser 1 5 10 15 LysVal Pro Gly Glu Asp Gly Thr Ser Ser Thr Ser Gly Met Leu Asp 20 25 30 LeuLeu Arg Asp Gln Val Asp Ser Leu Ser Ile Asn Asp Ser Thr Thr 35 40 45 GluPro Lys Thr Arg Leu Asp Pro Gly Leu Tyr Pro Trp Leu Lys Trp 50 55 60 ThrGlu Thr Ala Tyr Arg Ser Ser Thr Arg Ser Leu Ala Ser Thr Ile 65 70 75 80Val Met Gly Ala Leu Val Gln Gln Arg Gly Ser Gly Asn Gly Ile Thr 85 90 95Met Arg Glu Leu Glu Leu Ser Leu Gly Leu Asp Phe Thr Ser Glu Cys 100 105110 Asp Trp Leu Lys Thr Cys Tyr Val Asn Lys Asn Phe Val Phe Leu Ser 115120 125 Glu Lys Glu Ile Ala Val Asn Met Glu Val Glu Lys Phe Ile Cys Asn130 135 140 Glu Asn 145 16 736 DNA Infectious salmon anemia virus 16tgcaaagatt ggctatctac catgcatgag agaagcaaac ccaaaaccac gggagctgat 60cagacatgcc ttgaagaaga aaaagagacc agaggtggtt tacgcaatgg gagttcttct 120gacactgggg ggagagagcg gactgaccgt ggagtttcct gttccagaag gaaaaactgt 180gaaggtcaaa accttgaacc aattggtgaa cgggatgatc agtcgagcga cgatgaccct 240ctactgtgtg atgaaagatc caccatcggg aggcatggca acgctgatga gagaccacat 300caggaactgg ctgaaggagg aatcaggatg ccaggacgcg gatggtggag aggaaaaatg 360ggcaatggtg tatggtatga tttcacccga catggcagag gagaagacga tgctgaagga 420gctgaaaaca atgctacaca gcaggatgca gatgtatgct ctgggtgcaa gttcgaaagc 480cctagagaat ttagaaaagg ccatcgtcgc tgcagttcat cgacttccgg catcctgctc 540gacagagaag atggtgcttc tggggtacct gaagtaagct tcaaagaaag aatggaagcg 600gagaagaaga aactgaaaga gctggacgac aagatctaca agctaaggag aagattgagg 660aagatggagt acaagaaaat ggggatcaac cgagaaatcg acaaattgga agactctgta 720caataaaatc actagt 736 17 234 PRT Infectious salmon anemia virus 17 MetHis Glu Arg Ser Lys Pro Lys Thr Thr Gly Ala Asp Gln Thr Cys 1 5 10 15Leu Glu Glu Glu Lys Glu Thr Arg Gly Gly Leu Arg Asn Gly Ser Ser 20 25 30Ser Asp Thr Gly Gly Arg Glu Arg Thr Asp Arg Gly Val Ser Cys Ser 35 40 45Arg Arg Lys Asn Cys Glu Gly Gln Asn Leu Glu Pro Ile Gly Glu Arg 50 55 60Asp Asp Gln Ser Ser Asp Asp Asp Pro Leu Leu Cys Asp Glu Arg Ser 65 70 7580 Thr Ile Gly Arg His Gly Asn Ala Asp Glu Arg Pro His Gln Glu Leu 85 9095 Ala Glu Gly Gly Ile Arg Met Pro Gly Arg Gly Trp Trp Arg Gly Lys 100105 110 Met Gly Asn Gly Val Trp Tyr Asp Phe Thr Arg His Gly Arg Gly Glu115 120 125 Asp Asp Ala Glu Gly Ala Glu Asn Asn Ala Thr Gln Gln Asp AlaAsp 130 135 140 Val Cys Ser Gly Cys Lys Phe Glu Ser Pro Arg Glu Phe ArgLys Gly 145 150 155 160 His Arg Arg Cys Ser Ser Ser Thr Ser Gly Ile LeuLeu Asp Arg Glu 165 170 175 Asp Gly Ala Ser Gly Val Pro Glu Val Ser PheLys Glu Arg Met Glu 180 185 190 Ala Glu Lys Lys Lys Leu Lys Glu Leu AspAsp Lys Ile Tyr Lys Leu 195 200 205 Arg Arg Arg Leu Arg Lys Met Glu TyrLys Lys Met Gly Ile Asn Arg 210 215 220 Glu Ile Asp Lys Leu Glu Asp SerVal Gln 225 230 18 183 PRT Infectious salmon anemia virus 18 Met Arg GluAla Asn Pro Lys Pro Arg Glu Leu Ile Arg His Ala Leu 1 5 10 15 Lys LysLys Lys Arg Pro Glu Val Val Tyr Ala Met Gly Val Leu Leu 20 25 30 Thr LeuGly Gly Glu Ser Gly Leu Thr Val Glu Phe Pro Val Pro Glu 35 40 45 Gly LysThr Val Lys Val Lys Thr Leu Asn Gln Leu Val Asn Gly Met 50 55 60 Ile SerArg Ala Thr Met Thr Leu Tyr Cys Val Met Lys Asp Pro Pro 65 70 75 80 SerGly Gly Met Ala Thr Leu Met Arg Asp His Ile Arg Asn Trp Leu 85 90 95 LysGlu Glu Ser Gly Cys Gln Asp Ala Asp Gly Gly Glu Glu Lys Trp 100 105 110Ala Met Val Tyr Gly Met Ile Ser Pro Asp Met Ala Glu Glu Lys Thr 115 120125 Met Leu Lys Glu Leu Lys Thr Met Leu His Ser Arg Met Gln Met Tyr 130135 140 Ala Leu Gly Ala Ser Ser Lys Ala Leu Glu Asn Leu Glu Lys Ala Ile145 150 155 160 Val Ala Ala Val His Arg Leu Pro Ala Ser Cys Ser Thr GluLys Met 165 170 175 Val Leu Leu Gly Tyr Leu Lys 180 19 31 DNA ArtificialSequence Oligonucleotide Primer 19 aagcagtggt aacaacgcag agtagcaaag a 3120 21 DNA Artificial Sequence Oligonucleotide Primer 20 gaacgctctttaataaccat g 21 21 19 DNA Artificial Sequence Oligonucleotide Primer 21tcaaacatgc tttttcttc 19 22 19 DNA Artificial Sequence OligonucleotidePrimer 22 agcaaagatg gcacgattc 19 23 24 DNA Artificial SequenceOligonucleotide Primer 23 tgcacttttc tgtaaacgta caac 24 24 35 DNAArtificial Sequence Oligonucleotide Primer 24 aagcagtggt aacaacgcagagtctatcta ccatg 35 25 19 DNA Artificial Sequence Oligonucleotide Primer25 ttattgtaca gagtcttcc 19

1. (Amended) a pharmaceutical composition comprising a therapeuticallyeffective amount of an isolated nucleic acid molecule in apharmaceutically acceptable carrier, wherein the isolated nucleic acidmolecule [comprising] comprises at least one of (a) a nucleic acidsequence at least 70% identical to SEQ ID NO: 1; (b) a nucleic acidsequence at least 85% identical to SEQ ID NO: 3; [or] (c) a nucleic acidsequence at least 85% identical to SEQ ID NO: 11; (d) a nucleic acid atleast 80% identical to SEQ ID NO: 5, or (e) a nucleic acid at least 80%identical to SEQ ID NO: 9; and wherein the pharmaceutical compositioncan be used to induce an immune response to ISAV.
 2. (Amended) The[nucleic acid molecule] pharmaceutical composition according to claim[1] 34, wherein the nucleic acid sequence is at least 80% identical toSEQ ID NO: [1]
 5. 3. (Amended) The [nucleic acid molecule]pharmaceutical composition according to claim [1] 34, wherein thenucleic acid sequence is at least 90% identical to SEQ ID NO: [1] 5, SEQID NO: [3] 9, or SEQ ID NO:
 11. 4. (Amended) The [nucleic acid molecule]pharmaceutical composition according to claim [1] 34, wherein thenucleic acid sequence is at least 95% identical to SEQ ID NO: [1] 5, SEQID NO: [3] 9, or SEQ ID NO:
 11. 5. (Amended) The [nucleic acid molecule]pharmaceutical composition according to claim [1] 34, wherein thenucleic acid sequence consists essentially of SEQ ID NO: [1] 5, SEQ IDNO: [3] 9, or SEQ ID NO:
 11. 6. (Amended) The [nucleic acid molecule]pharmaceutical composition according to claim [1] 34, wherein thenucleic acid sequence is operably linked to a heterologous nucleic acidcomprising an expression control sequence.
 7. (Amended) The [nucleicacid molecule] pharmaceutical composition according to claim 6, whereinthe nucleic acid molecule encodes an antigenic epitope.
 8. (Reiterated)A vector comprising the nucleic acid molecule according to claim
 6. 9.(Reiterated) A host cell, comprising the nucleic acid according to claim6.
 10. (Reiterated) The host cell according to claim 9, wherein the cellis a fish cell.
 11. (Reiterated) The host cell according to claim 10,wherein the fish cell is from rainbow trout, coho salmon, chinooksalmon, amago salmon, chum salmon, sockeye salmon, Atlantic salmon,arctic char, brown trout, cutthroat trout, brook trout, catfish,tilapia, sea bream, seabass, flounder, or sturgeon.
 12. (Amended) [A] Anisolated nucleic acid comprising at least 100 consecutive nucleotides ofSEQ ID NO: [1]
 5. 13. (Amended) A transgenic animal, a nucleated cell ofwhich comprises: an expression control sequence operably linked to anucleic acid [sequence] molecule comprising (a) a nucleic acid sequenceat least 70% identical to SEQ ID NO: 1; (b) a nucleic acid sequence atleast 85% identical to SEQ ID NO: 3; [or] (c) a nucleic acid sequence atleast 85% identical to SEQ ID NO: 11; (d) a nucleic acid at least 80%identical to SEO ID NO: 5; or (e) a nucleic acid at least 80% identicalto SEQ ID NO: 9, and wherein the pharmaceutical composition can be usedto induce an immune response to ISAV; wherein the nucleic acid sequence[at least 70% identical to SEQ ID NO: 1, the nucleic acid sequence atleast 85% identical to SEQ ID NO: 3, or the nucleic acid sequence atleast 85% identical to SEQ ID NO: 11] encodes an antigenic epitope. 14.(Amended) The transgenic animal according to claim [13] 35, wherein theanimal exhibits an increased resistance to infection by infectioussalmon anemia virus as compared to a non-transformed animal of the samespecies.
 15. (Amended) The transgenic animal according to claim [13] 35,wherein the animal is an aquaculture animal.
 16. (Amended) Thetransgenic animal according to claim [15] 35, wherein the animal is afish.
 17. The transgenic animal according to claim 16, wherein the fishis rainbow trout, coho salmon, chinook salmon, amago salmon, chumsalmon, sockeye salmon, Atlantic salmon, arctic char, brown trout,cutthroat trout, brook trout, catfish, tilapia, sea bream, seabass,flounder, or sturgeon.
 18. A method of eliciting an immune responseagainst infections salmon anemia virus in a fish, comprising introducinginto the fish a therapeutically effective amount of the nucleic acidmolecule according to claim 6, wherein the nucleic acid molecule encodesan antigenic epitope of infectious salmon anemia virus, therebyeliciting an immune response against infectious salmon anemia virus inthe fish.
 19. (Amended) The method according to claim 18, wherein thenucleic acid molecule has a nucleic acid sequence at least 80% identicalto SEQ ID NO: [1]
 5. 20. (Amended) The method according to claim 19,wherein the nucleic acid molecule has a nucleic acid sequence at least85% identical to SEQ ID NO: [1]
 5. 21. (Amended) The method according toclaim 20, wherein the nucleic acid molecule has a nucleic acid sequenceat least 90% identical to SEQ ID NO: [1]
 5. 22. (Amended) The methodaccording to claim 21, wherein the nucleic acid molecule has a nucleicacid sequence at least 95% identical to SEQ ID NO: [1]
 5. 23. (Amended)The method according to claim 22, wherein the nucleic acid molecule hasa nucleic acid sequence consisting essentially of SEQ ID NO: [1]
 5. 24.(Amended) The method according to claim 18, wherein the nucleic acidsequence is at least 90% identical to SEQ ID NO: [3] 9 or SEQ ID NO: 11.25. (Amended) The method according to claim 24, wherein the nucleic acidsequence is at least 95% identical to SEQ ID NO: [3] 9 or SEQ ID NO: 11.26. (Amended) The method according to claim 25, wherein the nucleic acidsequence is at least 95% identical to SEQ ID NO: [3] 9 or SEQ ID NO: 11.27. (Amended) The method according to claim 26, wherein the nucleic acidsequence consists essentially of SEQ ID NO: [3] 9 or SEQ ID NO:
 11. 28.(Reiterated) A method of producing a transgenic fish, comprisingcontacting a nucleated cell of the fish with an amount of the nucleicacid molecule according to claim 6, wherein the amount of the nucleicacid molecule is sufficient to introduce the nucleic acid molecule intothe cell, thereby producing a transgenic fish.
 29. (Reiterated) Themethod according to claim 28, wherein the fish is rainbow trout, cohosalmon, chinook salmon, amago salmon, chum salmon, sockeye salmon,Atlantic salmon, arctic char, brown trout, cutthroat trout, brook trout,catfish, tilapia, sea bream, seabass, flounder, or sturgeon. 30.(Reiterated) A polypeptide encoded by the nucleic acid moleculeaccording to claim 1, or a conservative variant thereof. 31.(Reiterated) A method of inducing an immune response in a fish,comprising: delivering to the fish a therapeutically effective amount ofa composition comprising a polypeptide having an amino acid sequence asset forth as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17,or SEQ ID NO: 18, an antigenic fragment thereof, or a conservativevariant thereof; and wherein the polypeptide is an antigenic epitope ofinfectious salmon anemia virus, thereby eliciting an immune responseagainst infectious salmon anemia virus in the fish.
 32. (Reiterated) Themethod according to claim 31 wherein the polypeptide has an amino acidsequence consisting essentially of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 17, or SEQ ID NO:
 18. 33. (Reiterated) The methodaccording to claim 31 wherein the polypeptide comprises a fusionprotein.
 34. (New) The pharmaceutical composition of claim 1, comprisingat least one of (a) a nucleic acid sequence at least 85% identical toSEQ ID NO: 11, (b) a nucleic acid at least 80% identical to SEQ ID NO:5, and (c) a nucleic acid at least 80% identical to SEQ ID NO:
 9. 35.(New) A transgenic animal of claim 16, comprising an expression controlsequence operably linked to a nucleic acid molecule comprising at leastone of (a) a nucleic acid sequence at least 85% identical to SEQ ID NO:11, (b) a nucleic acid at least 80% identical to SEQ ID NO: 5, or (c) anucleic acid at least 80% identical to SEQ ID NO: 9, and wherein thepharmaceutical composition can be used to induce an immune response toISAV; wherein the nucleic acid sequence encodes an antigenic epitope.